LOCUS       sequence-147719-        6680 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Promoter: GAP6, minimal Promoter: GAP6. Figure 4. Expresses GFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6680)
  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 6680)
  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: PP262
FEATURES             Location/Qualifiers
     source          1..6680
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        157..568
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        576..623
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             642..1016
                     /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      1082..1329
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1098..1116)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(1404..1992)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1484..1503)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    2112..2161
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="attB site for the Mycopbacteriophage Bxb1 integrase 
                     (Xu et al., 2013)"
     protein_bind    3325..3343
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3346..3364
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             3434..3454
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4673..4822
                     /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             4829..4855
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      4897..5143
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     protein_bind    5655..5673
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5676..5694
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             join(5749..5751,5752..5754,5755..6465)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(5749..5751,5752..5754,5755..6465)
                     /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(5794..5815)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6055..6074)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6402..6423
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             6490..6519
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             6535..6552
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      join(6576..6680,1..142)
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
ORIGIN
        1 tcttctcgta cgagcttgct cctgatcagc ctatctcgca gctgatgaat atcttgtggt
       61 aggggtttgg gaaaatcatt cgagtttgat gtttttcttg gtatttccca ctcctcttca
      121 gagtacagaa gattaagtga gaccttcgtt tgtgcggatc ccccacacac catagcttca
      181 aaatgtttct actccttttt tactcttcca gattttctcg gactccgcgc atcgccgtac
      241 cacttcaaaa cacccaagca cagcatacta aattttccct ctttcttcct ctagggtgtc
      301 gttaattacc cgtactaaag gtttggaaaa gaaaaaagag accgcctcgt ttctttttct
      361 tcgtcgaaaa aggcaataaa aatttttatc acgtttcttt ttcttgaaat tttttttttt
      421 agtttttttc tctttcagtg acctccattg atatttaagt taataaacgg tcttcaattt
      481 ctcaagtttc agtttcattt ttcttgttct attacaactt tttttacttc ttgttcatta
      541 gaaagaaagc atagcaatct aatctaaggg gcggtgttga caattaatca tcggcatagt
      601 atatcggcat agtataatac gacaaggtga ggaactaaac catggccaag ttgaccagtg
      661 ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc
      721 tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga
      781 ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt
      841 gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc
      901 gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg
      961 ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgacacg
     1021 tccgacggcg gcccacgggt cccaggcctc ggagatccgt cccccttttc ctttgtcgat
     1081 atcatgtaat tagttatgtc acgcttacat tcacgccctc cccccacatc cgctctaacc
     1141 gaaaaggaag gagttagaca acctgaagtc taggtcccta tttatttttt tatagttatg
     1201 ttagtattaa gaacgttatt tatatttcaa atttttcttt tttttctgta cagacgcgtg
     1261 tacgcatgta acattatact gaaaaccttg cttgagaagg ttttgggacg ctcgaaggct
     1321 ttaatttgca agctggagac caacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     1381 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     1441 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     1501 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     1561 tccgcctttc tcccttcggg aagcgtggcg ctttctcaat gctcacgctg taggtatctc
     1621 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     1681 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     1741 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     1801 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     1861 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     1921 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     1981 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     2041 aactcacgtt aagggatttt ggtcatgaga tcagatctga gctcgtttgg ccgtggccgt
     2101 gctcgtcctc gtcggccggc ttgtcgacga cggcggtctc cgtcgtcagg atcatccggg
     2161 ccacaagctt gctgacagaa gcctcaagaa aaaaaaaatt cttcttcgac tatgctggag
     2221 gcagagatga tcgagccggt agttaactat atatagctaa attggttcca tcacctcgag
     2281 ttcaattctt gatttagtat acacataacc aaatttggat caagtttgaa gtaaaacttt
     2341 aacttcagct ccttacattt gcactaagat ctctgctact ctggtcccaa gtgaaccacc
     2401 ttttggaccc tattgaccgg accttaactt gccaaaccta aacgcttaat gcctcagacg
     2461 ttttaatgcc tctcaacacc tccaaggttg ctttcttgag catgcctact agggacttta
     2521 acgaactgtg gggttgcaga cagtttcagg cgtgtcccga ccaatatggc ctactagact
     2581 ctctgaaaaa tcacagtttt ccagtagttc cgatcaaatt accatcgaaa tggtcccata
     2641 aacggacatt tgacatccgt tcctgaatta tagtcttcca ccgtggatca tggtgttcct
     2701 ttttttccca aagaatatca gcatccctta actacgttag gtcagtgatg acaatggacc
     2761 aaattgttgc aaggtttttc tttttctttc atcggcacat ttcagcctca catgcgacta
     2821 ttatcgatca atgaaatcca tcaagattga aatcttaaaa ttgccccttt cacttgacag
     2881 gatccttttt tgtagaaatg tcttggtgtc ctcgtccaat caggtagcca tctctgaaat
     2941 atctggctcc gttgcaactc cgaacgacct gctggcaacg taaaattctc cggggtaaaa
     3001 ctttaatgtg gagtaatgga accagaaacg tctcttccct tctctctcct tccaccgccc
     3061 gttaccgtcc ctaggaaatt ttactctgct ggagagcttc ttctacggcc cccttgcagc
     3121 aatgctcttc ccagcattac gttgcgggta aaacggaggt cgtgtacccg acctagcagc
     3181 ccagggatgg aaaagtcccg gccgtcgctg gcaataatag cgggcggacg catgtcatga
     3241 gattattgga aaccaccaga atcgaatata aaaggcgaac acctttccca attttggttt
     3301 ctcctgaccc aaagacttta aatttcccta tcagtgatag agatctccct atcagtgata
     3361 gagataattt atttgtccct atttcaatca attgaacaac tatcaaaaca cactagtaaa
     3421 aatgcgcgga gctcctaaga aaaagcgcaa agtccggccg gcatctagac ccggggagcg
     3481 ccccttccag tgtcgcattt gcatgcggaa cttttcgcgc caggacaggc ttgacaggca
     3541 tacccgtact cataccggtg aaaaaccgtt tcagtgtcgg atctgtatgc gaaatttctc
     3601 ccagaaggag cacttggcgg ggcatctacg tacgcacacc ggcgagaagc cattccaatg
     3661 ccgaatatgc atgcgcaact tcagtcgccg cgacaacctg aaccggcacc taaaaaccca
     3721 cctgaggaac atatgcggcg gaggcacacc tgcagctgcg tcgactctag aggatccatc
     3781 tgctggagac atgagagctg ccaacctttg gccaagcccg ctcatgatca aacgctctaa
     3841 gaagaacagc ctggccttgt ccctgacggc cgaccagatg gtcagtgcct tgttggatgc
     3901 tgagcccccc atactctatt ccgagtatga tcctaccaga cccttcagtg aagcttcgat
     3961 gatgggctta ctgaccaacc tggcagacag ggagctggtt cacatgatca actgggcgaa
     4021 gagggtgcca ggctttgtgg atttgaccct ccatgatcag gtccaccttc tagaatgtgc
     4081 ctggctagag atcctgatga ttggtctcgt ctggcgctcc atggagcacc cagtgaagct
     4141 actgtttgct cctaacttgc tcttggacag gaaccaggga aaatgtgtag agggcatggt
     4201 ggagatcttc gacatgctgc tggctacatc atctcggttc cgcatgatga atctgcaggg
     4261 agaggagttt gtgtgcctca aatctattat tttgcttaat tctggagtgt acacatttct
     4321 gtccagcacc ctgaagtctc tggaagagaa ggaccatatc caccgagtcc tggacaagat
     4381 cacagacact ttgatccacc tgatggccaa ggcaggcctg accctgcagc agcagcacca
     4441 gcggctggcc cagctcctcc tcatcctctc ccacatcagg cacatgagta acaaaggcat
     4501 ggagcatctg tacagcatga agtgcaagaa cgtggtgccc ctctatgacc tgctgctgga
     4561 gatgctggac gcccaccgcc tacatgcgcc cactagccgt ggaggggcat ccgtggagga
     4621 gacggaccaa agccacttgg ccactgcggg ctctacttca tcgcctaggg ccgacgcgct
     4681 ggacgatttc gatctcgaca tgctgggttc tgatgccctc gatgactttg acctggatat
     4741 gttgggaagc gacgcattgg atgactttga tctggacatg ctcggctccg atgctctgga
     4801 cgatttcgat ctcgatatgt taattaacta cccgtacgac gttccggact acgcttcttg
     4861 aggtaccatc ggtagaccgg tcttgctaga ttctaatcaa gaggatgtca gaatgccatt
     4921 tgcctgagag atgcaggctt catttttgat acttttttat ttgtaaccta tatagtatag
     4981 gatttttttt gtcattttgt ttcttctcgt acgagcttgc tcctgatcag cctatctcgc
     5041 agctgatgaa tatcttgtgg taggggtttg ggaaaatcat tcgagtttga tgtttttctt
     5101 ggtatttccc actcctcttc agagtacaga agattaagtg agagctagca tatcatatag
     5161 aagtcatcgc ggcagatcaa ttcatcaatc ggagtgagga tattgcgagt ttaccaccat
     5221 caatcggagt gaggatctcc aattggtgac ggtccagtca tcaatcggag tgaggattca
     5281 gctgcttctc gaggccgcac atcaatcgga gtgaggatgt acagggtggg ctgttccacc
     5341 atcaatcgga gtgaggattt gcgtcaatgg ggcggagttc atcaatcgga gtgaggatat
     5401 cgaagtcatc gagagcactc atcaatcgga gtgaggatat actccaccca ttgacgtcac
     5461 atcaatcgga gtgaggattg gaaccagaag ggtctcttca tcaatcggag tgaggatacc
     5521 ctatgggcgc gccgtcgctg gcaataatag cgggcggacg catgtcatga gattattgga
     5581 aaccaccaga atcgaatata aaaggcgaac acctttccca attttggttt ctcctgaccc
     5641 aaagacttta aatttcccta tcagtgatag agatctccct atcagtgata gagataattt
     5701 atttgtccct atttcaatca attgaacaac tatcaaaaca cagaattcat ggtgagcaag
     5761 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     5821 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     5881 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     5941 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
     6001 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     6061 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     6121 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     6181 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
     6241 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     6301 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     6361 cagagcgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     6421 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaaggccgc ggcggccgcc
     6481 agctttctag aacaaaaact catctcagaa gaggatctga atagcgccgt cgaccatcat
     6541 catcatcatc attgaccggt cttgctagat tctaatcaag aggatgtcag aatgccattt
     6601 gcctgagaga tgcaggcttc atttttgata cttttttatt tgtaacctat atagtatagg
     6661 attttttttg tcattttgtt
//
