LOCUS       sequence-203186-        8012 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Methanol inducing hGH, and estrogen inducing G-CSF.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8012)
  AUTHORS   Cao J, Perez-Pinera P, Lowenhaupt K, Wu MR, Purcell O, de la 
            Fuente-Nunez C, Lu TK
  TITLE     Versatile and on-demand biologics co-production in yeast.
  JOURNAL   Nat Commun. 2018 Jan 8;9(1):77. doi: 10.1038/s41467-017-02587-w.
  PUBMED    29311542
REFERENCE   2  (bases 1 to 8012)
  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: "Nat 
            Commun."; date: "2018-01-8"; volume: "9(1):77. doi"; pages: " 
            10.1038/s41467-017-02587-w"
COMMENT     Sequence Label: JC034
FEATURES             Location/Qualifiers
     source          1..8012
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             14..43
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             59..76
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      100..346
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        361..774
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        781..828
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             847..1221
                     /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      1287..1534
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1303..1321)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     promoter        1549..2487
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     CDS             join(2488..2544,2545..2742)
                     /codon_start=1
                     /gene="MF-alpha-1"
                     /product="N-terminal secretion signal from S. cerevisiae 
                     alpha-factor"
                     /label=alpha-factor secretion signal
                     /note="Cleavage by the Kex2 protease occurs after the 
                     dibasic KR sequence."
                     /translation="MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLE
                     GDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR"
     terminator      3340..3586
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     rep_origin      complement(3647..4235)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3727..3746)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    4355..4404
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="attB site for the Mycopbacteriophage Bxb1 integrase 
                     (Xu et al., 2013)"
     promoter        4525..4931
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     CDS             4955..4975
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6194..6343
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             6350..6376
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      6418..6664
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             join(7225..7281,7282..7479)
                     /codon_start=1
                     /gene="MF-alpha-1"
                     /product="N-terminal secretion signal from S. cerevisiae 
                     alpha-factor"
                     /label=alpha-factor secretion signal
                     /note="Cleavage by the Kex2 protease occurs after the 
                     dibasic KR sequence."
                     /translation="MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLE
                     GDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR"
ORIGIN
        1 cgccagcttt ctagaacaaa aactcatctc agaagaggat ctgaatagcg ccgtcgacca
       61 tcatcatcat catcattgac cggtcttgct agattctaat caagaggatg tcagaatgcc
      121 atttgcctga gagatgcagg cttcattttt gatacttttt tatttgtaac ctatatagta
      181 taggattttt tttgtcattt tgtttcttct cgtacgagct tgctcctgat cagcctatct
      241 cgcagctgat gaatatcttg tggtaggggt ttgggaaaat cattcgagtt tgatgttttt
      301 cttggtattt cccactcctc ttcagagtac agaagattaa gtgagacctt cgtttgtgcg
      361 gatcccccac acaccatagc ttcaaaatgt ttctactcct tttttactct tccagatttt
      421 ctcggactcc gcgcatcgcc gtaccacttc aaaacaccca agcacagcat actaaatttc
      481 ccctctttct tcctctaggg tgtcgttaat tacccgtact aaaggtttgg aaaagaaaaa
      541 agagaccgcc tcgtttcttt ttcttcgtcg aaaaaggcaa taaaaatttt tatcacgttt
      601 ctttttcttg aaaatttttt tttttgattt ttttctcttt cgatgacctc ccattgatat
      661 ttaagttaat aaacggtctt caatttctca agtttcagtt tcatttttct tgttctatta
      721 caactttttt tacttcttgc tcattagaaa gaaagcatag caatctaatc taagggcggt
      781 gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa ggtgaggaac
      841 taaaccatgg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga
      901 gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga ggacgacttc
      961 gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg
     1021 ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg
     1081 tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac cgagatcggc
     1141 gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc
     1201 gtggccgagg agcaggactg acacgtccga cgcggcccga cgggtccgag gcctcggaga
     1261 tccgtccccc ttttcctttg tcgatatcat gtaattagtt atgtcacgct tacattcacg
     1321 ccctcccccc acatccgctc taaccgaaaa ggaaggagtt agacaacctg aagtctaggt
     1381 ccctatttat ttttttatag ttatgttagt attaagaacg ttatttatat ttcaaatttt
     1441 tctttttttt ctgtacagac gcgtgtacgc atgtaacatt atactgaaaa ccttgcttga
     1501 gaaggttttg ggacgctcga aggctttaat ttgcaagctg gagaccaaga tctaacatcc
     1561 aaagacgaaa ggttgaatga aacctttttg ccatccgaca tccacaggtc cattctcaca
     1621 cataagtgcc aaacgcaaca ggaggggata cactagcagc agaccgttgc aaacgcagga
     1681 cctccactcc tcttctcctc aacacccact tttgccatcg aaaaaccagc ccagttattg
     1741 ggcttgattg gagctcgctc attccaattc cttctattag gctactaaca ccatgacttt
     1801 attagcctgt ctatcctggc ccccctggcg aggttcatgt ttgtttattt ccgaatgcaa
     1861 caagctccgc attacacccg aacatcactc cagatgaggg ctttctgagt gtggggtcaa
     1921 atagtttcat gttccccaaa tggcccaaaa ctgacagttt aaacgctgtc ttggaaccta
     1981 atatgacaaa agcgtgatct catccaagat gaactaagtt tggttcgttg aaatgctaac
     2041 ggccagttgg tcaaaaagaa acttccaaaa gtcggcatac cgtttgtctt gtttggtatt
     2101 gattgacgaa tgctcaaaaa taatctcatt aatgcttagc gcagtctctc tatcgcttct
     2161 gaaccccggt gcacctgtgc cgaaacgcaa atggggaaac acccgctttt tggatgatta
     2221 tgcattgtct ccacattgta tgcttccaag attctggtgg gaatactgct gatagcctaa
     2281 cgttcatgat caaaatttaa ctgttctaac ccctacttga cagcaatata taaacagaag
     2341 gaagctgccc tgtcttaaac cttttttttt atcatcatta ttagcttact ttcataattg
     2401 cgactggttc caattgacaa gcttttgatt ttaacgactt ttaacgacaa cttgagaaga
     2461 tcaaaaaaca actaattatt cgaaacgatg agatttcctt caatttttac tgctgtttta
     2521 ttcgcagcat cctccgcatt agctgctcca gtcaacacta caacagaaga tgaaacggca
     2581 caaattccgg ctgaagctgt catcggttac tcagatttag aaggggattt cgatgttgct
     2641 gttttgccat tttccaacag cacaaataac gggttattgt ttataaatac tactattgcc
     2701 agcattgctg ctaaagaaga aggggtatct ctcgagaaga gattcccaac cattccctta
     2761 tctagacttt ttgacaacgc tatgctccgc gcccatcgtc tgcaccagct ggcctttgac
     2821 acctaccagg agtttgaaga agcctatatc ccaaaggaac agaagtattc attcctgcag
     2881 aacccccaga cctccctctg tttctcagag tctattccga caccctccaa cagggaggaa
     2941 acacaacaga aatccaacct agagctgctc cgcatctccc tgctgctcat ccagtcgtgg
     3001 ctggagcccg tgcagttcct caggagtgtc ttcgccaaca gcctggtgta cggcgcctct
     3061 gacagcaacg tctatgacct cctaaaggac ctagaggaag gcatccaaac gctgatgggg
     3121 aggctggaag atggcagccc ccggactggg cagatcttca agcagaccta cagcaagttc
     3181 gacacaaact cacacaacga tgacgcacta ctcaagaact acgggctgct ctactgcttc
     3241 aggaaggaca tggacaaggt cgagacattc ctgcgcatcg tgcagtgccg ctctgtggag
     3301 ggcagctgtg gcttctagac cggtcttgct agattctaat caagaggatg tcagaatgcc
     3361 atttgcctga gagatgcagg cttcattttt gatacttttt tatttgtaac ctatatagta
     3421 taggattttt tttgtcattt tgtttcttct cgtacgagct tgctcctgat cagcctatct
     3481 cgcagctgat gaatatcttg tggtaggggt ttgggaaaat cattcgagtt tgatgttttt
     3541 cttggtattt cccactcctc ttcagagtac agaagattaa gtgagacatg tgagcaaaag
     3601 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     3661 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     3721 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     3781 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     3841 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     3901 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     3961 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     4021 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     4081 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     4141 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     4201 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     4261 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agatcagatc
     4321 tgagctcgtt tggccgtggc cgtgctcgtc ctcgtcggcc ggcttgtcga cgacggcggt
     4381 ctccgtcgtc aggatcatcc gggccacaag cttgctgaca gaagcctcaa gaaaaaaaaa
     4441 attcttcttc gactatgctg gaggcagaga tgatcgagcc ggtagttaac tatatatagc
     4501 taaattggtt ccatcacctc gagtccacac accatagctt caaaatgttt ctactccttt
     4561 tttactcttc cagattttct cggactccgc gcatcgccgt accacttcaa aacacccaag
     4621 cacagcatac taaatttccc ctctttcttc ctctagggtg tcgttaatta cccgtactaa
     4681 aggtttggaa aagaaaaaag agaccgcctc gtttcttttt cttcgtcgaa aaaggcaata
     4741 aaaattttta tcacgtttct ttttcttgaa aatttttttt ttgatttttt tctctttcga
     4801 tgacctccca ttgatattta agttaataaa cggtcttcaa tttctcaagt ttcagtttca
     4861 tttttcttgt tctattacaa ctttttttac ttcttgctca ttagaaagaa agcatagcaa
     4921 tctaatctaa ggactagtaa aaatgcgcgg agctcctaag aaaaagcgca aagtccggcc
     4981 ggcatctaga cccggggagc gccccttcca gtgtcgcatt tgcatgcgga acttttcgcg
     5041 ccaggacagg cttgacaggc atacccgtac tcataccggt gaaaaaccgt ttcagtgtcg
     5101 gatctgtatg cgaaatttct cccagaagga gcacttggcg gggcatctac gtacgcacac
     5161 cggcgagaag ccattccaat gccgaatatg catgcgcaac ttcagtcgcc gcgacaacct
     5221 gaaccggcac ctaaaaaccc acctgaggaa catatgcggc ggaggcacac ctgcagctgc
     5281 gtcgactcta gaggatccat ctgctggaga catgagagct gccaaccttt ggccaagccc
     5341 gctcatgatc aaacgctcta agaagaacag cctggccttg tccctgacgg ccgaccagat
     5401 ggtcagtgcc ttgttggatg ctgagccccc catactctat tccgagtatg atcctaccag
     5461 acccttcagt gaagcttcga tgatgggctt actgaccaac ctggcagaca gggagctggt
     5521 tcacatgatc aactgggcga agagggtgcc aggctttgtg gatttgaccc tccatgatca
     5581 ggtccacctt ctagaatgtg cctggctaga gatcctgatg attggtctcg tctggcgctc
     5641 catggagcac ccagtgaagc tactgtttgc tcctaacttg ctcttggaca ggaaccaggg
     5701 aaaatgtgta gagggcatgg tggagatctt cgacatgctg ctggctacat catctcggtt
     5761 ccgcatgatg aatctgcagg gagaggagtt tgtgtgcctc aaatctatta ttttgcttaa
     5821 ttctggagtg tacacatttc tgtccagcac cctgaagtct ctggaagaga aggaccatat
     5881 ccaccgagtc ctggacaaga tcacagacac tttgatccac ctgatggcca aggcaggcct
     5941 gaccctgcag cagcagcacc agcggctggc ccagctcctc ctcatcctct cccacatcag
     6001 gcacatgagt aacaaaggca tggagcatct gtacagcatg aagtgcaaga acgtggtgcc
     6061 cctctatgac ctgctgctgg agatgctgga cgcccaccgc ctacatgcgc ccactagccg
     6121 tggaggggca tccgtggagg agacggacca aagccacttg gccactgcgg gctctacttc
     6181 atcgcctagg gccgacgcgc tggacgattt cgatctcgac atgctgggtt ctgatgccct
     6241 cgatgacttt gacctggata tgttgggaag cgacgcattg gatgactttg atctggacat
     6301 gctcggctcc gatgctctgg acgatttcga tctcgatatg ttaattaact acccgtacga
     6361 cgttccggac tacgcttctt gaggtaccat cggtagaccg gtcttgctag attctaatca
     6421 agaggatgtc agaatgccat ttgcctgaga gatgcaggct tcatttttga tactttttta
     6481 tttgtaacct atatagtata ggattttttt tgtcattttg tttcttctcg tacgagcttg
     6541 ctcctgatca gcctatctcg cagctgatga atatcttgtg gtaggggttt gggaaaatca
     6601 ttcgagtttg atgtttttct tggtatttcc cactcctctt cagagtacag aagattaagt
     6661 gagagctagc atatcatata gaagtcatcg cggcagatca attcatcaat cggagtgagg
     6721 atattgcgag tttaccacca tcaatcggag tgaggatctc caattggtga cggtccagtc
     6781 atcaatcgga gtgaggattc agctgcttct cgaggccgca catcaatcgg agtgaggatg
     6841 tacagggtgg gctgttccac catcaatcgg agtgaggatt tgcgtcaatg gggcggagtt
     6901 catcaatcgg agtgaggata tcgaagtcat cgagagcact catcaatcgg agtgaggata
     6961 tactccaccc attgacgtca catcaatcgg agtgaggatt ggaaccagaa gggtctcttc
     7021 atcaatcgga gtgaggatac cctatgggcg cgccgtcgct ggcaataata gcgggcggac
     7081 gcatgtcatg agattattgg aaaccaccag aatcgaatat aaaaggcgaa cacctttccc
     7141 aattttggtt tcccctgacc caaagacttt aaatttaatt tatttgtccc tatttcaatc
     7201 aattgaacaa ctatcaaaac acagatgaga tttccttcaa tttttactgc tgttttattc
     7261 gcagcatcct ccgcattagc tgctccagtc aacactacaa cagaagatga aacggcacaa
     7321 attccggctg aagctgtcat cggttactca gatttagaag gggatttcga tgttgctgtt
     7381 ttgccatttt ccaacagcac aaataacggg ttattgttta taaatactac tattgccagc
     7441 attgctgcta aagaagaagg ggtatctctc gagaagagaa tgactccttt gggtccagct
     7501 tcttccttgc ctcaatcctt cttgttgaag tgtttggagc aggttagaaa gatccagggt
     7561 gatggtgctg ctttgcaaga gaagttgtgt gctacttaca agttgtgtca cccagaagag
     7621 ttggttttgt tgggtcactc cttgggtatt ccttgggctc cattgtcctc ttgtccatcc
     7681 caagctttgc aattggctgg ttgtttgtcc caattgcact ccggtttgtt cttgtaccag
     7741 ggtttgttgc aagctttgga gggtatttct ccagagttgg gtccaacttt ggacacattg
     7801 cagttggacg ttgctgactt cgctactact atctggcaac agatggaaga attgggtatg
     7861 gctccagctt tgcagccaac tcaaggtgct atgccagctt ttgcttctgc tttccagaga
     7921 agagctggtg gtgttttggt tgcttctcac ttgcagtctt tcttggaggt ttcctacaga
     7981 gttttgagac acttggctca accataagcg gc
//
