LOCUS       sequence-174103-        4876 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of GFP in Pichia pastoris.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4876)
  AUTHORS   Yang J, Nie L, Chen B, Liu Y, Kong Y, Wang H, Diao L
  TITLE     Hygromycin-resistance vectors for gene expression in Pichia 
            pastoris.
  JOURNAL   Yeast. 2014 Apr;31(4):115-25.
  PUBMED    24822243
REFERENCE   2  (bases 1 to 4876)
  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: "Yeast."; 
            date: "2014-04"; volume: "31(4)"; pages: "115-25"
COMMENT     Sequence Label: pAHYB-GFP
FEATURES             Location/Qualifiers
     source          1..4876
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(116..704)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(196..215)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        790..1728
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     regulatory      1774..1783
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1780..1782,1783..1785,1786..2499)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1780..1782,1783..1785,1786..2496)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1825..1846)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2086..2105)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2433..2454
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2584..2613
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2629..2646
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2726..2972
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     gene            3031..4606
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        3031..3374
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             3375..4403
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             3375..4403
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     terminator      4409..4606
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     terminator      join(4670..4876,1..41)
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4686..4704)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
ORIGIN
        1 tgcttgagaa ggttttggga cgctcgaagg ctttaatttg caagctggag accaacatgt
       61 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
      121 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
      181 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
      241 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
      301 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
      361 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
      421 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
      481 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
      541 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
      601 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
      661 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
      721 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatgc
      781 atgagatcag atctaacatc caaagacgaa aggttgaatg aaaccttttt gccatccgac
      841 atccacaggt ccattctcac acataagtgc caaacgcaac aggaggggat acactagcag
      901 cagaccgttg caaacgcagg acctccactc ctcttctcct caacacccac ttttgccatc
      961 gaaaaaccag cccagttatt gggcttgatt ggagctcgct cattccaatt ccttctatta
     1021 ggctactaac accatgactt tattagcctg tctatcctgg cccccctggc gaggttcatg
     1081 tttgtttatt tccgaatgca acaagctccg cattacaccc gaacatcact ccagatgagg
     1141 gctttctgag tgtggggtca aatagtttca tgttccccaa atggcccaaa actgacagtt
     1201 taaacgctgt cttggaacct aatatgacaa aagcgtgatc tcatccaaga tgaactaagt
     1261 ttggttcgtt gaaatgctaa cggccagttg gtcaaaaaga aacttccaaa agtcggcata
     1321 ccgtttgtct tgtttggtat tgattgacga atgctcaaaa ataatctcat taatgcttag
     1381 cgcagtctct ctatcgcttc tgaaccccgg tgcacctgtg ccgaaacgca aatggggaaa
     1441 cacccgcttt ttggatgatt atgcattgtc tccacattgt atgcttccaa gattctggtg
     1501 ggaatactgc tgatagccta acgttcatga tcaaaattta actgttctaa cccctacttg
     1561 acagcaatat ataaacagaa ggaagctgcc ctgtcttaaa cctttttttt tatcatcatt
     1621 attagcttac tttcataatt gcgactggtt ccaattgaca agcttttgat tttaacgact
     1681 tttaacgaca acttgagaag atcaaaaaac aactaattat tcgaaacgag gaattcacgt
     1741 ggcccagccg gccgtctcgg atcggtaccg gtcgccacca tggtgagcaa gggcgaggag
     1801 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     1861 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     1921 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     1981 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     2041 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     2101 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     2161 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     2221 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     2281 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     2341 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
     2401 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     2461 gccgggatca ctctcggcat ggacgagctg tacaagtaaa gcggccgcga ctctagaatt
     2521 ccaactgagc gccggtcgct accattacca acttgtctgg tgtcaaaaat aataggccta
     2581 ctagaacaaa aactcatctc agaagaggat ctgaatagcg ccgtcgacca tcatcatcat
     2641 catcattgag tttgtagcct tagacatgac tgttcctcag ttcaagttgg gcacttacga
     2701 gaagaccggt cttgctagat tctaatcaag aggatgtcag aatgccattt gcctgagaga
     2761 tgcaggcttc atttttgata cttttttatt tgtaacctat atagtatagg attttttttg
     2821 tcattttgtt tcttctcgta cgagcttgct cctgatcagc ctatctcgca gctgatgaat
     2881 atcttgtggt aggggtttgg gaaaatcatt cgagtttgat gtttttcttg gtatttccca
     2941 ctcctcttca gagtacagaa gattaagtga gaccttcgtt tgtgcggatc tgtttagctt
     3001 gcctcgtccc cgccgggtca cccggccagc gacatggagg cccagaatac cctccttgac
     3061 agtcttgacg tgcgcagctc aggggcatga tgtgactgtc gcccgtacat ttagcccata
     3121 catccccatg tataatcatt tgcatccata cattttgatg gccgcacggc gcgaagcaaa
     3181 aattacggct cctcgctgcg gacctgcgag cagggaaacg ctcccctcac agacgcgttg
     3241 aattgtcccc acgccgcgcc cctgtagaga aatataaaag gttaggattt gccactgagg
     3301 ttcttctttc atatacttcc ttttaaaatc ttgctaggat acagttctca catcacatcc
     3361 gaacataaac aaccatgggt aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc
     3421 tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc gaagaatctc
     3481 gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg
     3541 atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg ctcccgattc
     3601 cggaagtgct tgacattggg gaattcagcg agagcctgac ctattgcatc tcccgccgtg
     3661 cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg
     3721 tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc gggttcggcc
     3781 cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata tgcgcgattg
     3841 ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt gcgtccgtcg
     3901 cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc cggcacctcg
     3961 tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata acagcggtca
     4021 ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac atcttcttct
     4081 ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg aggcatccgg
     4141 agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt gaccaactct
     4201 atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg
     4261 caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg
     4321 ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga cgccccagca
     4381 ctcgtccgag ggcaaaggaa taatcagtac tgacaataaa aagattcttg ttttcaagaa
     4441 cttgtcattt gtatagtttt tttatattgt agttgttcta ttttaatcaa atgttagcgt
     4501 gatttatatt ttttttcgcc tcgacatcat ctgcccagat gcgaagttaa gtgcgcagaa
     4561 agtaatatca tgcgtcaatc gtatgtgaat gctggtcgct atactgctgt cgattcgata
     4621 ctaacgccgc catccagtgt cgaaaacgag ctcgaattca tcgatgatat catgtaatta
     4681 gttatgtcac gcttacattc acgccctccc cccacatccg ctctaaccga aaaggaagga
     4741 gttagacaac ctgaagtcta ggtccctatt tattttttta tagttatgtt agtattaaga
     4801 acgttattta tatttcaaat ttttcttttt tttctgtaca gacgcgtgta cgcatgtaac
     4861 attatactga aaacct
//
