LOCUS       sequence-71975-e        5054 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FX cloning Pichia pastoris expression vector with AOX1 promoter and 
            N-terminal 10x His tag and 3C protease cleavage site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5054)
  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 5054)
  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: pICXNH3
FEATURES             Location/Qualifiers
     source          1..5054
                     /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             992..1021
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             1022..1045
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(1094..1399)
                     /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(1196..1215)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1741..2400)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2315..2334
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(2454..2460,2461..2478,2479..2484))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(2509..2633)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(2652..2672)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(2676..2696)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2736..2765
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2781..2798
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2878..3124
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        3139..3550
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        3558..3605
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3624..3998
                     /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      4064..4311
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4080..4098)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(4386..4974)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4466..4485)
                     /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 cattaaccat gcaccatcat catcaccatc atcatcatca
     1021 tttagaagtt ttgtttcaag gtccaagtag aagagcgagc tgcagactgg ctgtgtataa
     1081 gggagcctga catttatatt ccccagaaca tcaggttaat ggcgtttttg atgtcatttt
     1141 cgcggtggct gagatcagcc acttcttccc cgataacgga gaccggcaca ctggccatat
     1201 cggtggtcat catgcgccag ctttcatccc cgatatgcac caccgggtaa agttcacggg
     1261 agactttatc tgacagcaga cgtgcactgg ccagggggat caccatccgt cgcccgggcg
     1321 tgtcaataat atcactctgt acatccacaa acagacgata acggctctct cttttatagg
     1381 tgtaaacctt aaactgcatt tcaccagccc ctgttctcgt cagcaaaaga gccgttcatt
     1441 tcaataaacc gggcgacctc agccatccct tcctgatttt ccgctttcca gcgttcggca
     1501 cgcagacgac gggcttcatt ctgcatggtt gtgcttacca gaccggagat attgacatca
     1561 tatatgcctt gagcaactga tagctgtcgc tgtcaactgt cactgtaata cgctgcttca
     1621 tagcatacct ctttttgaca tacttcgggt atacatatca gtatatattc ttataccgca
     1681 aaaatcagcg cgcaaatacg catactgtta tctggctttt agtaagccgg atccacgcgt
     1741 ttacgccccg ccctgccact catcgcagta ctgttgtaat tcattaagca ttctgccgac
     1801 atggaagcca tcacaaacgg catgatgaac ctgaatcgcc agcggcatca gcaccttgtc
     1861 gccttgcgta taatatttgc ccatggtgaa aacgggggcg aagaagttgt ccatattggc
     1921 cacgtttaaa tcaaaactgg tgaaactcac ccagggattg gctgagacga aaaacatatt
     1981 ctcaataaac cctttaggga aataggccag gttttcaccg taacacgcca catcttgcga
     2041 atatatgtgt agaaactgcc ggaaatcgtc gtggtattca ctccagagcg atgaaaacgt
     2101 ttcagtttgc tcatggaaaa cggtgtaaca agggtgaaca ctatcccata tcaccagctc
     2161 accgtctttc attgccatac ggaattccgg atgagcattc atcaggcggg caagaatgtg
     2221 aataaaggcc ggataaaact tgtgcttatt tttctttacg gtctttaaaa aggccgtaat
     2281 atccagctga acggtctggt tataggtaca ttgagcaact gactgaaatg cctcaaaatg
     2341 ttctttacga tgccattggg atatatcaac ggtggtatat ccagtgattt ttttctccat
     2401 tttagcttcc ttagctcctg aaaatctcga cggatcctaa ctcaaaatcc acacattata
     2461 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gcggccgcca tagtgactgg
     2521 atatgttgtg ttttacagta ttatgtagtc tgttttttat gcaaaatcta atttaatata
     2581 ttgatattta tatcatttta cgtttctcgt tcagcttttt tgtacaaact tgtgatatcg
     2641 aattcgctag ctacctttct cttctttttt ggatctacct ttctcttctt ttttggggat
     2701 ccactagttc agctcttctg cataataact atctagaaca aaaactcatc tcagaagagg
     2761 atctgaatag cgccgtcgac catcatcatc atcatcattg agtttgtagc cttagacatg
     2821 actgttcctc agttcaagtt gggcacttac gagaagaccg gtcttgctag attctaatca
     2881 agaggatgtc agaatgccat ttgcctgaga gatgcaggct tcatttttga tactttttta
     2941 tttgtaacct atatagtata ggattttttt tgtcattttg tttcttctcg tacgagcttg
     3001 ctcctgatca gcctatctcg cagctgatga atatcttgtg gtaggggttt gggaaaatca
     3061 ttcgagtttg atgtttttct tggtatttcc cactcctctt cagagtacag aagattaagt
     3121 gagaccttcg tttgtgcgga tcccccacac accatagctt caaaatgttt ctactccttt
     3181 tttactcttc cagattttct cggactccgc gcatcgccgt accacttcaa aacacccaag
     3241 cacagcatac taaattttcc ctctttcttc ctctagggtg tcgttaatta cccgtactaa
     3301 aggtttggaa aagaaaaaag agaccgcctc gtttcttttt cttcgtcgaa aaaggcaata
     3361 aaaattttta tcacgtttct ttttcttgaa attttttttt ttagtttttt tctctttcag
     3421 tgacctccat tgatatttaa gttaataaac ggtcttcaat ttctcaagtt tcagtttcat
     3481 ttttcttgtt ctattacaac tttttttact tcttgttcat tagaaagaaa gcatagcaat
     3541 ctaatctaag gggcggtgtt gacaattaat catcggcata gtatatcggc atagtataat
     3601 acgacaaggt gaggaactaa accatggcca agttgaccag tgccgttccg gtgctcaccg
     3661 cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg gctcgggttc tcccgggact
     3721 tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt gaccctgttc atcagcgcgg
     3781 tccaggacca ggtggtgccg gacaacaccc tggcctgggt gtgggtgcgc ggcctggacg
     3841 agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt ccgggacgcc tccgggccgg
     3901 ccatgaccga gatcggcgag cagccgtggg ggcgggagtt cgccctgcgc gacccggccg
     3961 gcaactgcgt gcacttcgtg gccgaggagc aggactgaca cgtccgacgg cggcccacgg
     4021 gtcccaggcc tcggagatcc gtcccccttt tcctttgtcg atatcatgta attagttatg
     4081 tcacgcttac attcacgccc tccccccaca tccgctctaa ccgaaaagga aggagttaga
     4141 caacctgaag tctaggtccc tatttatttt tttatagtta tgttagtatt aagaacgtta
     4201 tttatatttc aaatttttct tttttttctg tacagacgcg tgtacgcatg taacattata
     4261 ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg ctttaatttg caagctggag
     4321 accaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     4381 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     4441 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     4501 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     4561 ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     4621 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     4681 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     4741 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     4801 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca
     4861 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     4921 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     4981 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     5041 ttggtcatga gatc
//
