LOCUS       pKSCTMF                6180 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Gene insertion/deletion in Pichia pastoris.
ACCESSION   .
VERSION     .
KEYWORDS    pKSCTMF
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6180)
  AUTHORS   Yang J, Jiang W, Yang S
  TITLE     mazF as a counter-selectable marker for unmarked genetic 
            modification of Pichia pastoris.
  JOURNAL   FEMS Yeast Res. 2009 Jun;9(4):600-9. doi: 
            10.1111/j.1567-1364.2009.00503.x. Epub 2009 Mar 25.
  PUBMED    19416369
REFERENCE   2  (bases 1 to 6180)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            //gt12365.cn
FEATURES             Location/Qualifiers
     source          1..6180
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     37..56
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     290..307
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    424..445
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        460..490
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    498..514
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     503..525
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     522..538
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     522..538
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     557..577
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        559..577
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     606..622
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     607..623
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      704..951
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     promoter        989..1927
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     CDS             2293..2322
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2338..2355
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2435..2681
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        2688..3095
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        3102..3149
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3168..3542
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     terminator      3608..3855
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3876..3892)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3876..3892)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3924..3943)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3925..3943)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3953..3970)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3953..3969)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3962..3984)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4111..4566
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4198..4217)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4408..4429
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4592..4696
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4697..5557
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4915..4934)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5728..136
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
       61 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
      121 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
      181 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
      241 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
      301 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
      361 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
      421 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgctcccggc
      481 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
      541 tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg gtaccgggcc
      601 ccccctcgag gtcgacggta tcgataagct tgattgacac gtccgacggc ggcccgacgg
      661 gtccgaggcc tcggagatcc gtcccccttt tcctttgtcg atatcatgta attagttatg
      721 tcacgcttac attcacgccc tccccccaca tccgctctaa ccgaaaagga aggagttaga
      781 caacctgaag tctaggtccc tatttatttt tttatagtta tgttagtatt aagaacgtta
      841 tttatatttc aaatttttct tttttttctg tacagacgcg tgtacgcatg taacattata
      901 ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg ctttaatttg caagctggag
      961 accaacaatc gaattcctgc agccctcaga tctaacatcc aaagacgaaa ggttgaatga
     1021 aacctttttg ccatccgaca tccacaggtc cattctcaca cataagtgcc aaacgcaaca
     1081 ggaggggata cactagcagc agaccgttgc aaacgcagga cctccactcc tcttctcctc
     1141 aacacccact tttgccatcg aaaaaccagc ccagttattg ggcttgattg gagctcgctc
     1201 attccaattc cttctattag gctactaaca ccatgacttt attagcctgt ctatcctggc
     1261 ccccctggcg aggttcatgt ttgtttattt ccgaatgcaa caagctccgc attacacccg
     1321 aacatcactc cagatgaggg ctttctgagt gtggggtcaa atagtttcat gttccccaaa
     1381 tggcccaaaa ctgacagttt aaacgctgtc ttggaaccta atatgacaaa agcgtgatct
     1441 catccaagat gaactaagtt tggttcgttg aaatgctaac ggccagttgg tcaaaaagaa
     1501 acttccaaaa gtcggcatac cgtttgtctt gtttggtatt gattgacgaa tgctcaaaaa
     1561 taatctcatt aatgcttagc gcagtctctc tatcgcttct gaaccccggt gcacctgtgc
     1621 cgaaacgcaa atggggaaac acccgctttt tggatgatta tgcattgtct ccacattgta
     1681 tgcttccaag attctggtgg gaatactgct gatagcctaa cgttcatgat caaaatttaa
     1741 ctgttctaac ccctacttga cagcaatata taaacagaag gaagctgccc tgtcttaaac
     1801 cttttttttt atcatcatta ttagcttact ttcataattg cgactggttc caattgacaa
     1861 gcttttgatt ttaacgactt ttaacgacaa cttgagaaga tcaaaaaaca actaattatt
     1921 cgatacgatg gtaagccgat acgtacccga tatgggcgat ctgatttggg ttgattttga
     1981 cccgacaaaa ggtagcgagc aagctggaca tcgtccagct gttgtcctga gtcctttcat
     2041 gtacaacaac aaaacaggta tgtgtctgtg tgttccttgt acaacgcaat caaaaggata
     2101 tccgttcgaa gttgttttat ccggtcagga acgtgatggc gtagcgttag ctgatcaggt
     2161 aaaaagtatc gcctggcggg caagaggagc aacgaagaaa ggaacagttg ccccagagga
     2221 attacaactc attaaagcca aaattaacgt actgattggg taggtcgagc cgcggcggcc
     2281 gccagctttc tagaacaaaa actcatctca gaagaggatc tgaatagcgc cgtcgaccat
     2341 catcatcatc atcattgagt ttgtagcctt agacatgact gttcctcagt tcaagttggg
     2401 cacttacgag aagaccggtc ttgctagatt ctaatcaaga ggatgtcaga atgccatttg
     2461 cctgagagat gcaggcttca tttttgatac ttttttattt gtaacctata tagtatagga
     2521 ttttttttgt cattttgttt cttctcgtac gagcttgctc ctgatcagcc tatctcgcag
     2581 ctgatgaata tcttgtggta ggggtttggg aaaatcattc gagtttgatg tttttcttgg
     2641 tatttcccac tcctcttcag agtacagaag attaagtgag agatccccca cacaccatag
     2701 cttcaaaatg tttctactcc ttttttactc ttccagattt tctcggactc cgcgcatcgc
     2761 cgtaccactt caaaacaccc aagcacagca tactaaattt cccctctttc ttcctctagg
     2821 gtgtcgttaa ttacccgtac taaaggtttg gaaaagaaaa aagagaccgc ctcgtttctt
     2881 tttcttcgtc gaaaaaggca ataaaaattt ttatcacgtt tctttttctt gaaaattttt
     2941 ttttttgatt tttttctctt tcgatgacct cccattgata tttaagttaa taaacggtct
     3001 tcaatttctc aagtttcagt ttcatttttc ttgttctatt acaacttttt ttacttcttg
     3061 ctcattagaa agaaagcata gcaatctaat ctaagggcgg tgttgacaat taatcatcgg
     3121 catagtatat cggcatagta taatacgaca aggtgaggaa ctaaaccatg gccaagttga
     3181 ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg
     3241 accggctcgg gttctcccgg gacttcgtgg aggacgactt cgccggtgtg gtccgggacg
     3301 acgtgaccct gttcatcagc gcggtccagg accaggtggt gccggacaac accctggcct
     3361 gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc gtgtccacga
     3421 acttccggga cgcctccggg ccggccatga ccgagatcgg cgagcagccg tgggggcggg
     3481 agttcgccct gcgcgacccg gccggcaact gcgtgcactt cgtggccgag gagcaggact
     3541 gacacgtccg acgcggcccg acgggtccga ggcctcggag atccgtcccc cttttccttt
     3601 gtcgatatca tgtaattagt tatgtcacgc ttacattcac gccctccccc cacatccgct
     3661 ctaaccgaaa aggaaggagt tagacaacct gaagtctagg tccctattta tttttttata
     3721 gttatgttag tattaagaac gttatttata tttcaaattt ttcttttttt tctgtacaga
     3781 cgcgtgtacg catgtaacat tatactgaaa accttgcttg agaaggtttt gggacgctcg
     3841 aaggctttaa tttgcaagct ggagaccaac agggggatcc actagttcta gagcggccgc
     3901 caccgcggtg gagctccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg
     3961 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     4021 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     4081 aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
     4141 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     4201 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     4261 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     4321 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     4381 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     4441 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     4501 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     4561 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     4621 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     4681 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     4741 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     4801 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     4861 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     4921 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     4981 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     5041 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     5101 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     5161 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     5221 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     5281 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     5341 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     5401 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     5461 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     5521 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     5581 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     5641 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     5701 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     5761 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     5821 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     5881 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     5941 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     6001 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     6061 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     6121 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
//