LOCUS       sequence-71979-e        5770 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FX cloning Pichia pastoris expression vector with AOX1 promoter and 
            C-terminal 3C protease cleavage site, GFP and 10x His tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5770)
  AUTHORS   Geertsma ER, Dutzler R
  TITLE     A versatile and efficient high-throughput cloning tool for 
            structural biology.
  JOURNAL   Biochemistry. 2011 Apr 19;50(15):3272-8. Epub 2011 Mar 25.
  PUBMED    21410291
REFERENCE   2  (bases 1 to 5770)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Biochemistry."; date: "2011-04-19"; volume: "50(15)"; pages: 
            "3272-8. Epub 2011 Mar 25"
COMMENT     Sequence Label: pICXC3GH
FEATURES             Location/Qualifiers
     source          1..5770
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2..940
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     CDS             complement(1039..1344)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(1141..1160)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1686..2345)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2260..2279
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(2399..2405,2406..2423,2424..2429))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(2454..2578)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(2597..2617)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(2621..2641)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2668..2691
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2698..3408
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     CDS             3415..3444
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             3452..3481
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3497..3514
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      3594..3840
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        3855..4266
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        4274..4321
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4340..4714
                     /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      4780..5027
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4796..4814)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(5102..5690)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5182..5201)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag
       61 gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt
      121 tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc
      181 agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta
      241 acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta
      301 tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg
      361 agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct
      421 gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg
      481 ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggca taccgtttgt
      541 cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct
      601 ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct
      661 ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact
      721 gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat
      781 atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt
      841 actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga
      901 caacttgaga agatcaaaaa acaactaatt attcgaaacg aggaattcac gtggcccagc
      961 cggccgtctc ggatcggtac cttaaccatg agtagaagag cgagctgcag actggctgtg
     1021 tataagggag cctgacattt atattcccca gaacatcagg ttaatggcgt ttttgatgtc
     1081 attttcgcgg tggctgagat cagccacttc ttccccgata acggagaccg gcacactggc
     1141 catatcggtg gtcatcatgc gccagctttc atccccgata tgcaccaccg ggtaaagttc
     1201 acgggagact ttatctgaca gcagacgtgc actggccagg gggatcacca tccgtcgccc
     1261 gggcgtgtca ataatatcac tctgtacatc cacaaacaga cgataacggc tctctctttt
     1321 ataggtgtaa accttaaact gcatttcacc agcccctgtt ctcgtcagca aaagagccgt
     1381 tcatttcaat aaaccgggcg acctcagcca tcccttcctg attttccgct ttccagcgtt
     1441 cggcacgcag acgacgggct tcattctgca tggttgtgct taccagaccg gagatattga
     1501 catcatatat gccttgagca actgatagct gtcgctgtca actgtcactg taatacgctg
     1561 cttcatagca tacctctttt tgacatactt cgggtataca tatcagtata tattcttata
     1621 ccgcaaaaat cagcgcgcaa atacgcatac tgttatctgg cttttagtaa gccggatcca
     1681 cgcgtttacg ccccgccctg ccactcatcg cagtactgtt gtaattcatt aagcattctg
     1741 ccgacatgga agccatcaca aacggcatga tgaacctgaa tcgccagcgg catcagcacc
     1801 ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata
     1861 ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg gattggctga gacgaaaaac
     1921 atattctcaa taaacccttt agggaaatag gccaggtttt caccgtaaca cgccacatct
     1981 tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa
     2041 aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc ccatatcacc
     2101 agctcaccgt ctttcattgc catacggaat tccggatgag cattcatcag gcgggcaaga
     2161 atgtgaataa aggccggata aaacttgtgc ttatttttct ttacggtctt taaaaaggcc
     2221 gtaatatcca gctgaacggt ctggttatag gtacattgag caactgactg aaatgcctca
     2281 aaatgttctt tacgatgcca ttgggatata tcaacggtgg tatatccagt gatttttttc
     2341 tccattttag cttccttagc tcctgaaaat ctcgacggat cctaactcaa aatccacaca
     2401 ttatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgcggc cgccatagtg
     2461 actggatatg ttgtgtttta cagtattatg tagtctgttt tttatgcaaa atctaattta
     2521 atatattgat atttatatca ttttacgttt ctcgttcagc ttttttgtac aaacttgtga
     2581 tatcgaattc gctagctacc tttctcttct tttttggatc tacctttctc ttcttttttg
     2641 gggatccact agttcagctc ttctgcatta gaagttttgt ttcaaggtcc acaattctct
     2701 aaaggagaag aattattcac tggtgttgtc ccaattttgg ttgaattaga tggtgatgtt
     2761 aatggtcaca aattttctgt ctccggtgaa ggtgaaggtg atgctactta cggtaaattg
     2821 accttaaaat ttatttgtac tactggtaaa ttgccagttc catggccaac cttagtcact
     2881 actttcggtt atggtgttca atgttttgcg agatacccag atcatatgaa acaacatgac
     2941 tttttcaagt ctgccatgcc agaaggttat gttcaagaaa gaactatttt tttcaaagat
     3001 gacggtaact acaagaccag agctgaagtc aagtttgaag gtgatacctt agttaataga
     3061 atcgaattaa aaggtattga ttttaaagaa gatggtaaca ttttaggtca caaattggaa
     3121 tacaactata actctcacaa tgtttacatc atggctgaca aacaaaagaa tggtatcaaa
     3181 gttaacttca aaattagaca caacattgaa gatggttctg ttcaattagc tgaccattat
     3241 caacaaaata ctccaattgg tgatggtcca gtcttgttac cagacaacca ttacttatcc
     3301 actcaatctg ccttatccaa agatccaaac gaaaagagag accacatggt cttgttagaa
     3361 tttgttactg ctgctggtat tacccatggt atggatgaat tgtacaaaac tagtcatcat
     3421 catcaccatc atcatcacca tcattaatct agaacaaaaa ctcatctcag aagaggatct
     3481 gaatagcgcc gtcgaccatc atcatcatca tcattgagtt tgtagcctta gacatgactg
     3541 ttcctcagtt caagttgggc acttacgaga agaccggtct tgctagattc taatcaagag
     3601 gatgtcagaa tgccatttgc ctgagagatg caggcttcat ttttgatact tttttatttg
     3661 taacctatat agtataggat tttttttgtc attttgtttc ttctcgtacg agcttgctcc
     3721 tgatcagcct atctcgcagc tgatgaatat cttgtggtag gggtttggga aaatcattcg
     3781 agtttgatgt ttttcttggt atttcccact cctcttcaga gtacagaaga ttaagtgaga
     3841 ccttcgtttg tgcggatccc ccacacacca tagcttcaaa atgtttctac tcctttttta
     3901 ctcttccaga ttttctcgga ctccgcgcat cgccgtacca cttcaaaaca cccaagcaca
     3961 gcatactaaa ttttccctct ttcttcctct agggtgtcgt taattacccg tactaaaggt
     4021 ttggaaaaga aaaaagagac cgcctcgttt ctttttcttc gtcgaaaaag gcaataaaaa
     4081 tttttatcac gtttcttttt cttgaaattt ttttttttag tttttttctc tttcagtgac
     4141 ctccattgat atttaagtta ataaacggtc ttcaatttct caagtttcag tttcattttt
     4201 cttgttctat tacaactttt tttacttctt gttcattaga aagaaagcat agcaatctaa
     4261 tctaaggggc ggtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga
     4321 caaggtgagg aactaaacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg
     4381 cgacgtcgcc ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt
     4441 ggaggacgac ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca
     4501 ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct
     4561 gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat
     4621 gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa
     4681 ctgcgtgcac ttcgtggccg aggagcagga ctgacacgtc cgacggcggc ccacgggtcc
     4741 caggcctcgg agatccgtcc cccttttcct ttgtcgatat catgtaatta gttatgtcac
     4801 gcttacattc acgccctccc cccacatccg ctctaaccga aaaggaagga gttagacaac
     4861 ctgaagtcta ggtccctatt tattttttta tagttatgtt agtattaaga acgttattta
     4921 tatttcaaat ttttcttttt tttctgtaca gacgcgtgta cgcatgtaac attatactga
     4981 aaaccttgct tgagaaggtt ttgggacgct cgaaggcttt aatttgcaag ctggagacca
     5041 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     5101 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     5161 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     5221 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     5281 gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     5341 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     5401 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     5461 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     5521 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     5581 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     5641 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     5701 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     5761 tcatgagatc
//
