LOCUS       sequence-71977-e        5764 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FX cloning Pichia pastoris expression vector with AOX1 promoter and 
            N-terminal 10x His tag, GFP and 3C protease cleavage site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5764)
  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 5764)
  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: pICXNHG3
FEATURES             Location/Qualifiers
     source          1..5764
                     /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             985..1014
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             1015..1725
                     /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             1732..1755
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(1804..2109)
                     /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(1906..1925)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(2451..3110)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3025..3044
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(3164..3170,3171..3188,3189..3194))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(3219..3343)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(3362..3382)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(3386..3406)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3446..3475
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3491..3508
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      3588..3834
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        3849..4260
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        4268..4315
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4334..4708
                     /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      4774..5021
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4790..4808)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(5096..5684)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5176..5195)
                     /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 catgcaccat catcatcacc atcatcatca tcattctaaa
     1021 ggagaagaat tattcactgg tgttgtccca attttggttg aattagatgg tgatgttaat
     1081 ggtcacaaat tttctgtctc cggtgaaggt gaaggtgatg ctacttacgg taaattgacc
     1141 ttaaaattta tttgtactac tggtaaattg ccagttccat ggccaacctt agtcactact
     1201 ttcggttatg gtgttcaatg ttttgcgaga tacccagatc atatgaaaca acatgacttt
     1261 ttcaagtctg ccatgccaga aggttatgtt caagaaagaa ctattttttt caaagatgac
     1321 ggtaactaca agaccagagc tgaagtcaag tttgaaggtg ataccttagt taatagaatc
     1381 gaattaaaag gtattgattt taaagaagat ggtaacattt taggtcacaa attggaatac
     1441 aactataact ctcacaatgt ttacatcatg gctgacaaac aaaagaatgg tatcaaagtt
     1501 aacttcaaaa ttagacacaa cattgaagat ggttctgttc aattagctga ccattatcaa
     1561 caaaatactc caattggtga tggtccagtc ttgttaccag acaaccatta cttatccact
     1621 caatctgcct tatccaaaga tccaaacgaa aagagagacc acatggtctt gttagaattt
     1681 gttactgctg ctggtattac ccatggtatg gatgaattgt acaaaactag tttagaagtt
     1741 ttgtttcaag gtccaagtag aagagcgagc tgcagactgg ctgtgtataa gggagcctga
     1801 catttatatt ccccagaaca tcaggttaat ggcgtttttg atgtcatttt cgcggtggct
     1861 gagatcagcc acttcttccc cgataacgga gaccggcaca ctggccatat cggtggtcat
     1921 catgcgccag ctttcatccc cgatatgcac caccgggtaa agttcacggg agactttatc
     1981 tgacagcaga cgtgcactgg ccagggggat caccatccgt cgcccgggcg tgtcaataat
     2041 atcactctgt acatccacaa acagacgata acggctctct cttttatagg tgtaaacctt
     2101 aaactgcatt tcaccagccc ctgttctcgt cagcaaaaga gccgttcatt tcaataaacc
     2161 gggcgacctc agccatccct tcctgatttt ccgctttcca gcgttcggca cgcagacgac
     2221 gggcttcatt ctgcatggtt gtgcttacca gaccggagat attgacatca tatatgcctt
     2281 gagcaactga tagctgtcgc tgtcaactgt cactgtaata cgctgcttca tagcatacct
     2341 ctttttgaca tacttcgggt atacatatca gtatatattc ttataccgca aaaatcagcg
     2401 cgcaaatacg catactgtta tctggctttt agtaagccgg atccacgcgt ttacgccccg
     2461 ccctgccact catcgcagta ctgttgtaat tcattaagca ttctgccgac atggaagcca
     2521 tcacaaacgg catgatgaac ctgaatcgcc agcggcatca gcaccttgtc gccttgcgta
     2581 taatatttgc ccatggtgaa aacgggggcg aagaagttgt ccatattggc cacgtttaaa
     2641 tcaaaactgg tgaaactcac ccagggattg gctgagacga aaaacatatt ctcaataaac
     2701 cctttaggga aataggccag gttttcaccg taacacgcca catcttgcga atatatgtgt
     2761 agaaactgcc ggaaatcgtc gtggtattca ctccagagcg atgaaaacgt ttcagtttgc
     2821 tcatggaaaa cggtgtaaca agggtgaaca ctatcccata tcaccagctc accgtctttc
     2881 attgccatac ggaattccgg atgagcattc atcaggcggg caagaatgtg aataaaggcc
     2941 ggataaaact tgtgcttatt tttctttacg gtctttaaaa aggccgtaat atccagctga
     3001 acggtctggt tataggtaca ttgagcaact gactgaaatg cctcaaaatg ttctttacga
     3061 tgccattggg atatatcaac ggtggtatat ccagtgattt ttttctccat tttagcttcc
     3121 ttagctcctg aaaatctcga cggatcctaa ctcaaaatcc acacattata cgagccggaa
     3181 gcataaagtg taaagcctgg ggtgcctaat gcggccgcca tagtgactgg atatgttgtg
     3241 ttttacagta ttatgtagtc tgttttttat gcaaaatcta atttaatata ttgatattta
     3301 tatcatttta cgtttctcgt tcagcttttt tgtacaaact tgtgatatcg aattcgctag
     3361 ctacctttct cttctttttt ggatctacct ttctcttctt ttttggggat ccactagttc
     3421 agctcttctg cataataact atctagaaca aaaactcatc tcagaagagg atctgaatag
     3481 cgccgtcgac catcatcatc atcatcattg agtttgtagc cttagacatg actgttcctc
     3541 agttcaagtt gggcacttac gagaagaccg gtcttgctag attctaatca agaggatgtc
     3601 agaatgccat ttgcctgaga gatgcaggct tcatttttga tactttttta tttgtaacct
     3661 atatagtata ggattttttt tgtcattttg tttcttctcg tacgagcttg ctcctgatca
     3721 gcctatctcg cagctgatga atatcttgtg gtaggggttt gggaaaatca ttcgagtttg
     3781 atgtttttct tggtatttcc cactcctctt cagagtacag aagattaagt gagaccttcg
     3841 tttgtgcgga tcccccacac accatagctt caaaatgttt ctactccttt tttactcttc
     3901 cagattttct cggactccgc gcatcgccgt accacttcaa aacacccaag cacagcatac
     3961 taaattttcc ctctttcttc ctctagggtg tcgttaatta cccgtactaa aggtttggaa
     4021 aagaaaaaag agaccgcctc gtttcttttt cttcgtcgaa aaaggcaata aaaattttta
     4081 tcacgtttct ttttcttgaa attttttttt ttagtttttt tctctttcag tgacctccat
     4141 tgatatttaa gttaataaac ggtcttcaat ttctcaagtt tcagtttcat ttttcttgtt
     4201 ctattacaac tttttttact tcttgttcat tagaaagaaa gcatagcaat ctaatctaag
     4261 gggcggtgtt gacaattaat catcggcata gtatatcggc atagtataat acgacaaggt
     4321 gaggaactaa accatggcca agttgaccag tgccgttccg gtgctcaccg cgcgcgacgt
     4381 cgccggagcg gtcgagttct ggaccgaccg gctcgggttc tcccgggact tcgtggagga
     4441 cgacttcgcc ggtgtggtcc gggacgacgt gaccctgttc atcagcgcgg tccaggacca
     4501 ggtggtgccg gacaacaccc tggcctgggt gtgggtgcgc ggcctggacg agctgtacgc
     4561 cgagtggtcg gaggtcgtgt ccacgaactt ccgggacgcc tccgggccgg ccatgaccga
     4621 gatcggcgag cagccgtggg ggcgggagtt cgccctgcgc gacccggccg gcaactgcgt
     4681 gcacttcgtg gccgaggagc aggactgaca cgtccgacgg cggcccacgg gtcccaggcc
     4741 tcggagatcc gtcccccttt tcctttgtcg atatcatgta attagttatg tcacgcttac
     4801 attcacgccc tccccccaca tccgctctaa ccgaaaagga aggagttaga caacctgaag
     4861 tctaggtccc tatttatttt tttatagtta tgttagtatt aagaacgtta tttatatttc
     4921 aaatttttct tttttttctg tacagacgcg tgtacgcatg taacattata ctgaaaacct
     4981 tgcttgagaa ggttttggga cgctcgaagg ctttaatttg caagctggag accaacatgt
     5041 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     5101 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     5161 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     5221 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     5281 cgctttctca atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     5341 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     5401 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     5461 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     5521 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     5581 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     5641 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     5701 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     5761 gatc
//
