LOCUS       sequence-147715-        6651 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Promoter: GAP6, minimal Promoter: GCW14. Figure 4. Expresses GFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6651)
  AUTHORS   Perez-Pinera P, Han N, Cleto S, Cao J, Purcell O, Shah KA, Lee K, 
            Ram R, Lu TK
  TITLE     Synthetic biology and microbioreactor platforms for programmable 
            production of biologics at the point-of-care.
  JOURNAL   Nat Commun. 2016 Jul 29;7:12211. doi: 10.1038/ncomms12211.
  PUBMED    27470089
REFERENCE   2  (bases 1 to 6651)
  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: "2016-07-29"; volume: "7:12211. doi"; pages: " 
            10.1038/ncomms12211"
COMMENT     Sequence Label: PP276
FEATURES             Location/Qualifiers
     source          1..6651
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      422..669
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(438..456)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(744..1332)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(824..843)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    1452..1501
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="attB site for the Mycopbacteriophage Bxb1 integrase 
                     (Xu et al., 2013)"
     protein_bind    2665..2683
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2686..2704
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             2774..2794
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4013..4162
                     /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             4169..4195
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      4237..4483
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             join(5060..5062,5063..5065,5066..5776)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(5060..5062,5063..5065,5066..5776)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5105..5126)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5366..5385)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5713..5734
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5801..5830
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             5846..5863
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      5887..6133
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        6148..6559
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        6567..6614
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             join(6633..6651,1..356)
                     /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"
ORIGIN
        1 ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc
       61 tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga
      121 ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt
      181 gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc
      241 gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg
      301 ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgacacg
      361 tccgacggcg gcccacgggt cccaggcctc ggagatccgt cccccttttc ctttgtcgat
      421 atcatgtaat tagttatgtc acgcttacat tcacgccctc cccccacatc cgctctaacc
      481 gaaaaggaag gagttagaca acctgaagtc taggtcccta tttatttttt tatagttatg
      541 ttagtattaa gaacgttatt tatatttcaa atttttcttt tttttctgta cagacgcgtg
      601 tacgcatgta acattatact gaaaaccttg cttgagaagg ttttgggacg ctcgaaggct
      661 ttaatttgca agctggagac caacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
      721 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
      781 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
      841 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
      901 tccgcctttc tcccttcggg aagcgtggcg ctttctcaat gctcacgctg taggtatctc
      961 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     1021 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     1081 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     1141 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     1201 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     1261 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     1321 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     1381 aactcacgtt aagggatttt ggtcatgaga tcagatctga gctcgtttgg ccgtggccgt
     1441 gctcgtcctc gtcggccggc ttgtcgacga cggcggtctc cgtcgtcagg atcatccggg
     1501 ccacaagctt gctgacagaa gcctcaagaa aaaaaaaatt cttcttcgac tatgctggag
     1561 gcagagatga tcgagccggt agttaactat atatagctaa attggttcca tcacctcgag
     1621 ttcaattctt gatttagtat acacataacc aaatttggat caagtttgaa gtaaaacttt
     1681 aacttcagct ccttacattt gcactaagat ctctgctact ctggtcccaa gtgaaccacc
     1741 ttttggaccc tattgaccgg accttaactt gccaaaccta aacgcttaat gcctcagacg
     1801 ttttaatgcc tctcaacacc tccaaggttg ctttcttgag catgcctact agggacttta
     1861 acgaactgtg gggttgcaga cagtttcagg cgtgtcccga ccaatatggc ctactagact
     1921 ctctgaaaaa tcacagtttt ccagtagttc cgatcaaatt accatcgaaa tggtcccata
     1981 aacggacatt tgacatccgt tcctgaatta tagtcttcca ccgtggatca tggtgttcct
     2041 ttttttccca aagaatatca gcatccctta actacgttag gtcagtgatg acaatggacc
     2101 aaattgttgc aaggtttttc tttttctttc atcggcacat ttcagcctca catgcgacta
     2161 ttatcgatca atgaaatcca tcaagattga aatcttaaaa ttgccccttt cacttgacag
     2221 gatccttttt tgtagaaatg tcttggtgtc ctcgtccaat caggtagcca tctctgaaat
     2281 atctggctcc gttgcaactc cgaacgacct gctggcaacg taaaattctc cggggtaaaa
     2341 ctttaatgtg gagtaatgga accagaaacg tctcttccct tctctctcct tccaccgccc
     2401 gttaccgtcc ctaggaaatt ttactctgct ggagagcttc ttctacggcc cccttgcagc
     2461 aatgctcttc ccagcattac gttgcgggta aaacggaggt cgtgtacccg acctagcagc
     2521 ccagggatgg aaaagtcccg gccgtcgctg gcaataatag cgggcggacg catgtcatga
     2581 gattattgga aaccaccaga atcgaatata aaaggcgaac acctttccca attttggttt
     2641 ctcctgaccc aaagacttta aatttcccta tcagtgatag agatctccct atcagtgata
     2701 gagataattt atttgtccct atttcaatca attgaacaac tatcaaaaca cactagtaaa
     2761 aatgcgcgga gctcctaaga aaaagcgcaa agtccggccg gcatctagac ccggggagcg
     2821 ccccttccag tgtcgcattt gcatgcggaa cttttcgcgc caggacaggc ttgacaggca
     2881 tacccgtact cataccggtg aaaaaccgtt tcagtgtcgg atctgtatgc gaaatttctc
     2941 ccagaaggag cacttggcgg ggcatctacg tacgcacacc ggcgagaagc cattccaatg
     3001 ccgaatatgc atgcgcaact tcagtcgccg cgacaacctg aaccggcacc taaaaaccca
     3061 cctgaggaac atatgcggcg gaggcacacc tgcagctgcg tcgactctag aggatccatc
     3121 tgctggagac atgagagctg ccaacctttg gccaagcccg ctcatgatca aacgctctaa
     3181 gaagaacagc ctggccttgt ccctgacggc cgaccagatg gtcagtgcct tgttggatgc
     3241 tgagcccccc atactctatt ccgagtatga tcctaccaga cccttcagtg aagcttcgat
     3301 gatgggctta ctgaccaacc tggcagacag ggagctggtt cacatgatca actgggcgaa
     3361 gagggtgcca ggctttgtgg atttgaccct ccatgatcag gtccaccttc tagaatgtgc
     3421 ctggctagag atcctgatga ttggtctcgt ctggcgctcc atggagcacc cagtgaagct
     3481 actgtttgct cctaacttgc tcttggacag gaaccaggga aaatgtgtag agggcatggt
     3541 ggagatcttc gacatgctgc tggctacatc atctcggttc cgcatgatga atctgcaggg
     3601 agaggagttt gtgtgcctca aatctattat tttgcttaat tctggagtgt acacatttct
     3661 gtccagcacc ctgaagtctc tggaagagaa ggaccatatc caccgagtcc tggacaagat
     3721 cacagacact ttgatccacc tgatggccaa ggcaggcctg accctgcagc agcagcacca
     3781 gcggctggcc cagctcctcc tcatcctctc ccacatcagg cacatgagta acaaaggcat
     3841 ggagcatctg tacagcatga agtgcaagaa cgtggtgccc ctctatgacc tgctgctgga
     3901 gatgctggac gcccaccgcc tacatgcgcc cactagccgt ggaggggcat ccgtggagga
     3961 gacggaccaa agccacttgg ccactgcggg ctctacttca tcgcctaggg ccgacgcgct
     4021 ggacgatttc gatctcgaca tgctgggttc tgatgccctc gatgactttg acctggatat
     4081 gttgggaagc gacgcattgg atgactttga tctggacatg ctcggctccg atgctctgga
     4141 cgatttcgat ctcgatatgt taattaacta cccgtacgac gttccggact acgcttcttg
     4201 aggtaccatc ggtagaccgg tcttgctaga ttctaatcaa gaggatgtca gaatgccatt
     4261 tgcctgagag atgcaggctt catttttgat acttttttat ttgtaaccta tatagtatag
     4321 gatttttttt gtcattttgt ttcttctcgt acgagcttgc tcctgatcag cctatctcgc
     4381 agctgatgaa tatcttgtgg taggggtttg ggaaaatcat tcgagtttga tgtttttctt
     4441 ggtatttccc actcctcttc agagtacaga agattaagtg agagctagca tatcatatag
     4501 aagtcatcgc ggcagatcaa ttcatcaatc ggagtgagga tattgcgagt ttaccaccat
     4561 caatcggagt gaggatctcc aattggtgac ggtccagtca tcaatcggag tgaggattca
     4621 gctgcttctc gaggccgcac atcaatcgga gtgaggatgt acagggtggg ctgttccacc
     4681 atcaatcgga gtgaggattt gcgtcaatgg ggcggagttc atcaatcgga gtgaggatat
     4741 cgaagtcatc gagagcactc atcaatcgga gtgaggatat actccaccca ttgacgtcac
     4801 atcaatcgga gtgaggattg gaaccagaag ggtctcttca tcaatcggag tgaggatacc
     4861 ctatgggcgc gcctagggca gtttgagttt aaaataggaa atataaacaa atataccgcg
     4921 aaaaaggttt gtttatagct tttcgcctgg tgccgtacgg tataaataca tactctcctc
     4981 ccccccctgg ttctcttttt cttttgttac ttacatttta ccgttccgtc actcgcttca
     5041 ctcaacaaca aaagaattca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     5101 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     5161 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     5221 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
     5281 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     5341 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     5401 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     5461 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     5521 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     5581 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     5641 gctgctgccc gacaaccact acctgagcac ccagagcgcc ctgagcaaag accccaacga
     5701 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     5761 ggacgagctg tacaaggccg cggcggccgc cagctttcta gaacaaaaac tcatctcaga
     5821 agaggatctg aatagcgccg tcgaccatca tcatcatcat cattgaccgg tcttgctaga
     5881 ttctaatcaa gaggatgtca gaatgccatt tgcctgagag atgcaggctt catttttgat
     5941 acttttttat ttgtaaccta tatagtatag gatttttttt gtcattttgt ttcttctcgt
     6001 acgagcttgc tcctgatcag cctatctcgc agctgatgaa tatcttgtgg taggggtttg
     6061 ggaaaatcat tcgagtttga tgtttttctt ggtatttccc actcctcttc agagtacaga
     6121 agattaagtg agaccttcgt ttgtgcggat cccccacaca ccatagcttc aaaatgtttc
     6181 tactcctttt ttactcttcc agattttctc ggactccgcg catcgccgta ccacttcaaa
     6241 acacccaagc acagcatact aaattttccc tctttcttcc tctagggtgt cgttaattac
     6301 ccgtactaaa ggtttggaaa agaaaaaaga gaccgcctcg tttctttttc ttcgtcgaaa
     6361 aaggcaataa aaatttttat cacgtttctt tttcttgaaa tttttttttt tagttttttt
     6421 ctctttcagt gacctccatt gatatttaag ttaataaacg gtcttcaatt tctcaagttt
     6481 cagtttcatt tttcttgttc tattacaact ttttttactt cttgttcatt agaaagaaag
     6541 catagcaatc taatctaagg ggcggtgttg acaattaatc atcggcatag tatatcggca
     6601 tagtataata cgacaaggtg aggaactaaa ccatggccaa gttgaccagt g
//
