LOCUS       sequence-147713-        6612 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Promoter: GAP, minimal Promoter: GCW14. Figure 4. Expresses GFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6612)
  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 6612)
  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: PP275
FEATURES             Location/Qualifiers
     source          1..6612
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      424..671
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(440..458)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(746..1334)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(826..845)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    1454..1503
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="attB site for the Mycopbacteriophage Bxb1 integrase 
                     (Xu et al., 2013)"
     promoter        2228..2704
                     /label=GAP promoter
                     /note="promoter for Pichia pastoris 
                     glyceraldehyde-3-phosphate dehydrogenase"
     CDS             2735..2755
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3974..4123
                     /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             4130..4156
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      4198..4444
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             join(5023..5025,5026..5028,5029..5739)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(5023..5025,5026..5028,5029..5739)
                     /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(5068..5089)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5329..5348)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5676..5697
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5764..5793
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             5809..5826
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      5850..6096
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        6111..6522
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        6530..6577
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             join(6596..6612,1..358)
                     /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 tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg
       61 gctcgggttc tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt
      121 gaccctgttc atcagcgcgg tccaggacca ggtggtgccg gacaacaccc tggcctgggt
      181 gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt
      241 ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg ggcgggagtt
      301 cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc aggactgaca
      361 cgtccgacgg cggcccacgg gtcccaggcc tcggagatcc gtcccccttt tcctttgtcg
      421 atatcatgta attagttatg tcacgcttac attcacgccc tccccccaca tccgctctaa
      481 ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt tttatagtta
      541 tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg tacagacgcg
      601 tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg
      661 ctttaatttg caagctggag accaacatgt gagcaaaagg ccagcaaaag gccaggaacc
      721 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
      781 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
      841 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
      901 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc
      961 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     1021 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     1081 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     1141 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     1201 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     1261 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     1321 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     1381 aaaactcacg ttaagggatt ttggtcatga gatcagatct gagctcgttt ggccgtggcc
     1441 gtgctcgtcc tcgtcggccg gcttgtcgac gacggcggtc tccgtcgtca ggatcatccg
     1501 ggccacaagc ttgctgacag aagcctcaag aaaaaaaaaa ttcttcttcg actatgctgg
     1561 aggcagagat gatcgagccg gtagttaact atatatagct aaattggttc catcacctcg
     1621 agtcaattct tgatttagta tacacataac caaatttgga tcaagtttga agtaaaactt
     1681 taacttcagc tccttacatt tgcactaaga tctctgctac tctggtccca agtgaaccac
     1741 cttttggacc ctattgaccg gaccttaact tgccaaacct aaacgcttaa tgcctcagac
     1801 gttttaatgc ctctcaacac ctccaaggtt gctttcttga gcatgcctac tagggacttt
     1861 aacgaactgt ggggttgcag acagtttcag gcgtgtcccg accaatatgg cctactagac
     1921 tctctgaaaa atcacagttt tccagtagtt ccgatcaaat taccatcgaa atggtcccat
     1981 aaacggacat ttgacatccg ttcctgaatt atagtcttcc accgtggatc atggtgttcc
     2041 tttttttccc aaagaatatc agcatccctt aactacgtta ggtcagtgat gacaatggac
     2101 caaattgttg caaggttttt ctttttcttt catcggcaca tttcagcctc acatgcgact
     2161 attatcgatc aatgaaatcc atcaagattg aaatcttaaa attgcccctt tcacttgaca
     2221 ggatcctttt ttgtagaaat gtcttggtgt cctcgtccaa tcaggtagcc atctctgaaa
     2281 tatctggctc cgttgcaact ccgaacgacc tgctggcaac gtaaaattct ccggggtaaa
     2341 actttaatgt ggagtaatgg aaccagaaac gtctcttccc ttctctctcc ttccaccgcc
     2401 cgttaccgtc cctaggaaat tttactctgc tggagagctt cttctacggc ccccttgcag
     2461 caatgctctt cccagcatta cgttgcgggt aaaacggagg tcgtgtaccc gacctagcag
     2521 cccagggatg gaaaagtccc ggccgtcgct ggcaataata gcgggcggac gcatgtcatg
     2581 agattattgg aaaccaccag aatcgaatat aaaaggcgaa cacctttccc aattttggtt
     2641 tcccctgacc caaagacttt aaatttaatt tatttgtccc tatttcaatc aattgaacaa
     2701 ctatcaaaac acactagtaa aaatgcgcgg agctcctaag aaaaagcgca aagtccggcc
     2761 ggcatctaga cccggggagc gccccttcca gtgtcgcatt tgcatgcgga acttttcgcg
     2821 ccaggacagg cttgacaggc atacccgtac tcataccggt gaaaaaccgt ttcagtgtcg
     2881 gatctgtatg cgaaatttct cccagaagga gcacttggcg gggcatctac gtacgcacac
     2941 cggcgagaag ccattccaat gccgaatatg catgcgcaac ttcagtcgcc gcgacaacct
     3001 gaaccggcac ctaaaaaccc acctgaggaa catatgcggc ggaggcacac ctgcagctgc
     3061 gtcgactcta gaggatccat ctgctggaga catgagagct gccaaccttt ggccaagccc
     3121 gctcatgatc aaacgctcta agaagaacag cctggccttg tccctgacgg ccgaccagat
     3181 ggtcagtgcc ttgttggatg ctgagccccc catactctat tccgagtatg atcctaccag
     3241 acccttcagt gaagcttcga tgatgggctt actgaccaac ctggcagaca gggagctggt
     3301 tcacatgatc aactgggcga agagggtgcc aggctttgtg gatttgaccc tccatgatca
     3361 ggtccacctt ctagaatgtg cctggctaga gatcctgatg attggtctcg tctggcgctc
     3421 catggagcac ccagtgaagc tactgtttgc tcctaacttg ctcttggaca ggaaccaggg
     3481 aaaatgtgta gagggcatgg tggagatctt cgacatgctg ctggctacat catctcggtt
     3541 ccgcatgatg aatctgcagg gagaggagtt tgtgtgcctc aaatctatta ttttgcttaa
     3601 ttctggagtg tacacatttc tgtccagcac cctgaagtct ctggaagaga aggaccatat
     3661 ccaccgagtc ctggacaaga tcacagacac tttgatccac ctgatggcca aggcaggcct
     3721 gaccctgcag cagcagcacc agcggctggc ccagctcctc ctcatcctct cccacatcag
     3781 gcacatgagt aacaaaggca tggagcatct gtacagcatg aagtgcaaga acgtggtgcc
     3841 cctctatgac ctgctgctgg agatgctgga cgcccaccgc ctacatgcgc ccactagccg
     3901 tggaggggca tccgtggagg agacggacca aagccacttg gccactgcgg gctctacttc
     3961 atcgcctagg gccgacgcgc tggacgattt cgatctcgac atgctgggtt ctgatgccct
     4021 cgatgacttt gacctggata tgttgggaag cgacgcattg gatgactttg atctggacat
     4081 gctcggctcc gatgctctgg acgatttcga tctcgatatg ttaattaact acccgtacga
     4141 cgttccggac tacgcttctt gaggtaccat cggtagaccg gtcttgctag attctaatca
     4201 agaggatgtc agaatgccat ttgcctgaga gatgcaggct tcatttttga tactttttta
     4261 tttgtaacct atatagtata ggattttttt tgtcattttg tttcttctcg tacgagcttg
     4321 ctcctgatca gcctatctcg cagctgatga atatcttgtg gtaggggttt gggaaaatca
     4381 ttcgagtttg atgtttttct tggtatttcc cactcctctt cagagtacag aagattaagt
     4441 gagagctagc atatcatata gaagtcatcg cggcagatca attcatcaat cggagtgagg
     4501 atattgcgag tttaccacca tcaatcggag tgaggatctc caattggtga cggtccagtc
     4561 atcaatcgga gtgaggattc agctgcttct cgaggccgca catcaatcgg agtgaggatg
     4621 tacagggtgg gctgttccac catcaatcgg agtgaggatt tgcgtcaatg gggcggagtt
     4681 catcaatcgg agtgaggata tcgaagtcat cgagagcact catcaatcgg agtgaggata
     4741 tactccaccc attgacgtca catcaatcgg agtgaggatt ggaaccagaa gggtctcttc
     4801 atcaatcgga gtgaggatac cctatgggcg cgcctagggc agattttgag tttaaaatag
     4861 gaaatataaa caaatatacc gcgaaaaagg tttgtttata gcttttcgcc tggtgccgta
     4921 cggtataaat acatactctc ctcccccccc ttgttctctt tttcttttgt tacttacatt
     4981 ttaccgttcc gtcactcgct tcactcaaca acaaaagaat tcatggtgag caagggcgag
     5041 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
     5101 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag
     5161 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc
     5221 tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag
     5281 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
     5341 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
     5401 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac
     5461 aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc
     5521 aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
     5581 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagagc
     5641 gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     5701 gccgccggga tcactctcgg catggacgag ctgtacaagg ccgcggcggc cgccagcttt
     5761 ctagaacaaa aactcatctc agaagaggat ctgaatagcg ccgtcgacca tcatcatcat
     5821 catcattgac cggtcttgct agattctaat caagaggatg tcagaatgcc atttgcctga
     5881 gagatgcagg cttcattttt gatacttttt tatttgtaac ctatatagta taggattttt
     5941 tttgtcattt tgtttcttct cgtacgagct tgctcctgat cagcctatct cgcagctgat
     6001 gaatatcttg tggtaggggt ttgggaaaat cattcgagtt tgatgttttt cttggtattt
     6061 cccactcctc ttcagagtac agaagattaa gtgagacctt cgtttgtgcg gatcccccac
     6121 acaccatagc ttcaaaatgt ttctactcct tttttactct tccagatttt ctcggactcc
     6181 gcgcatcgcc gtaccacttc aaaacaccca agcacagcat actaaatttt ccctctttct
     6241 tcctctaggg tgtcgttaat tacccgtact aaaggtttgg aaaagaaaaa agagaccgcc
     6301 tcgtttcttt ttcttcgtcg aaaaaggcaa taaaaatttt tatcacgttt ctttttcttg
     6361 aaattttttt ttttagtttt tttctctttc agtgacctcc attgatattt aagttaataa
     6421 acggtcttca atttctcaag tttcagtttc atttttcttg ttctattaca acttttttta
     6481 cttcttgttc attagaaaga aagcatagca atctaatcta aggggcggtg ttgacaatta
     6541 atcatcggca tagtatatcg gcatagtata atacgacaag gtgaggaact aaaccatggc
     6601 caagttgacc ag
//
