LOCUS       sequence-147716-        6166 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Promoter: ppTEF1, minimal Promoter: GCW14. Figure 4. Expresses GFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6166)
  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 6166)
  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: PP273
FEATURES             Location/Qualifiers
     source          1..6166
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      420..667
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(436..454)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(742..1330)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(822..841)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    1450..1499
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="attB site for the Mycopbacteriophage Bxb1 integrase 
                     (Xu et al., 2013)"
     CDS             2285..2305
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3524..3673
                     /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             3680..3706
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      3748..3994
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             join(4573..4575,4576..4578,4579..5289)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4573..4575,4576..4578,4579..5289)
                     /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(4618..4639)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4879..4898)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5226..5247
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5314..5343
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             5359..5376
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      5400..5646
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        5661..6072
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        6080..6127
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             join(6146..6166,1..354)
                     /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 gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac cgaccggctc
       61 gggttctccc gggacttcgt ggaggacgac ttcgccggtg tggtccggga cgacgtgacc
      121 ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg
      181 gtgcgcggcc tggacgagct gtacgccgag tggtcggagg tcgtgtccac gaacttccgg
      241 gacgcctccg ggccggccat gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc
      301 ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga ctgacacgtc
      361 cgacggcggc ccacgggtcc caggcctcgg agatccgtcc cccttttcct ttgtcgatat
      421 catgtaatta gttatgtcac gcttacattc acgccctccc cccacatccg ctctaaccga
      481 aaaggaagga gttagacaac ctgaagtcta ggtccctatt tattttttta tagttatgtt
      541 agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca gacgcgtgta
      601 cgcatgtaac attatactga aaaccttgct tgagaaggtt ttgggacgct cgaaggcttt
      661 aatttgcaag ctggagacca acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
      721 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
      781 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
      841 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
      901 cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag
      961 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     1021 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     1081 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     1141 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg
     1201 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     1261 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     1321 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     1381 ctcacgttaa gggattttgg tcatgagatc agatctgagc tcgtttggcc gtggccgtgc
     1441 tcgtcctcgt cggccggctt gtcgacgacg gcggtctccg tcgtcaggat catccgggcc
     1501 acaagcttgc tgacagaagc ctcaagaaaa aaaaaattct tcttcgacta tgctggaggc
     1561 agagatgatc gagccggtag ttaactatat atagctaaat tggttccatc acctcgagtc
     1621 agcatctggt tacgtaactc tggcaaccag taacacgctt aaggtttgga acaacactaa
     1681 actaccttgc ggtactacca ttgacactac acatccttaa ttccaatcct gtctggcctc
     1741 cttcaccttt taaccatctt gcccattcca actcgtgtca gattgcgtat caagtgaaaa
     1801 aaaaaaaatt ttaaaatctt taacccaatc aggtaataac tgtcgcctct tttatctgcc
     1861 gcactgcatg aggtgtcccc ttagtgggaa agagtactga gccaaccctg gaggacagca
     1921 agggaaaaat acctacaact tgcttcataa tggtcgtaaa aacaatcctt gtcggatata
     1981 agtgttgtag actgtccctt atcctctgcg atgttcttcc tctcaaagtt tgcgatttct
     2041 ctctatcaga attgccatca agagactcag gactaatttc gcagtcccac acgcactcgt
     2101 acatgattgg ctgaaatttc cctaaagaat ttctttttca cgaaaatttt tttttacaca
     2161 agattttcag cagatataaa atggagagca ggacctccgc tgtgactctt cttttttttc
     2221 ttttattctc actacataca ttttagttat tcgccaacgg caactagtaa aaatgcgcgg
     2281 agctcctaag aaaaagcgca aagtccggcc ggcatctaga cccggggagc gccccttcca
     2341 gtgtcgcatt tgcatgcgga acttttcgcg ccaggacagg cttgacaggc atacccgtac
     2401 tcataccggt gaaaaaccgt ttcagtgtcg gatctgtatg cgaaatttct cccagaagga
     2461 gcacttggcg gggcatctac gtacgcacac cggcgagaag ccattccaat gccgaatatg
     2521 catgcgcaac ttcagtcgcc gcgacaacct gaaccggcac ctaaaaaccc acctgaggaa
     2581 catatgcggc ggaggcacac ctgcagctgc gtcgactcta gaggatccat ctgctggaga
     2641 catgagagct gccaaccttt ggccaagccc gctcatgatc aaacgctcta agaagaacag
     2701 cctggccttg tccctgacgg ccgaccagat ggtcagtgcc ttgttggatg ctgagccccc
     2761 catactctat tccgagtatg atcctaccag acccttcagt gaagcttcga tgatgggctt
     2821 actgaccaac ctggcagaca gggagctggt tcacatgatc aactgggcga agagggtgcc
     2881 aggctttgtg gatttgaccc tccatgatca ggtccacctt ctagaatgtg cctggctaga
     2941 gatcctgatg attggtctcg tctggcgctc catggagcac ccagtgaagc tactgtttgc
     3001 tcctaacttg ctcttggaca ggaaccaggg aaaatgtgta gagggcatgg tggagatctt
     3061 cgacatgctg ctggctacat catctcggtt ccgcatgatg aatctgcagg gagaggagtt
     3121 tgtgtgcctc aaatctatta ttttgcttaa ttctggagtg tacacatttc tgtccagcac
     3181 cctgaagtct ctggaagaga aggaccatat ccaccgagtc ctggacaaga tcacagacac
     3241 tttgatccac ctgatggcca aggcaggcct gaccctgcag cagcagcacc agcggctggc
     3301 ccagctcctc ctcatcctct cccacatcag gcacatgagt aacaaaggca tggagcatct
     3361 gtacagcatg aagtgcaaga acgtggtgcc cctctatgac ctgctgctgg agatgctgga
     3421 cgcccaccgc ctacatgcgc ccactagccg tggaggggca tccgtggagg agacggacca
     3481 aagccacttg gccactgcgg gctctacttc atcgcctagg gccgacgcgc tggacgattt
     3541 cgatctcgac atgctgggtt ctgatgccct cgatgacttt gacctggata tgttgggaag
     3601 cgacgcattg gatgactttg atctggacat gctcggctcc gatgctctgg acgatttcga
     3661 tctcgatatg ttaattaact acccgtacga cgttccggac tacgcttctt gaggtaccat
     3721 cggtagaccg gtcttgctag attctaatca agaggatgtc agaatgccat ttgcctgaga
     3781 gatgcaggct tcatttttga tactttttta tttgtaacct atatagtata ggattttttt
     3841 tgtcattttg tttcttctcg tacgagcttg ctcctgatca gcctatctcg cagctgatga
     3901 atatcttgtg gtaggggttt gggaaaatca ttcgagtttg atgtttttct tggtatttcc
     3961 cactcctctt cagagtacag aagattaagt gagagctagc atatcatata gaagtcatcg
     4021 cggcagatca attcatcaat cggagtgagg atattgcgag tttaccacca tcaatcggag
     4081 tgaggatctc caattggtga cggtccagtc atcaatcgga gtgaggattc agctgcttct
     4141 cgaggccgca catcaatcgg agtgaggatg tacagggtgg gctgttccac catcaatcgg
     4201 agtgaggatt tgcgtcaatg gggcggagtt catcaatcgg agtgaggata tcgaagtcat
     4261 cgagagcact catcaatcgg agtgaggata tactccaccc attgacgtca catcaatcgg
     4321 agtgaggatt ggaaccagaa gggtctcttc atcaatcgga gtgaggatac cctatgggcg
     4381 cgcctagggc agattttgag tttaaaatag gaaatataaa caaatatacc gcgaaaaagg
     4441 tttgtttata gcttttcgcc tggtgccgta cggtataaat acatactctc ctcccccccc
     4501 tggttctctt tttcttttgt tacttacatt ttaccgttcc gtcactcgct tcactcaaca
     4561 acaaaagaat tcatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     4621 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
     4681 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     4741 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
     4801 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     4861 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     4921 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     4981 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     5041 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     5101 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     5161 cccgacaacc actacctgag cacccagagc gccctgagca aagaccccaa cgagaagcgc
     5221 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     5281 ctgtacaagg ccgcggcggc cgccagcttt ctagaacaaa aactcatctc agaagaggat
     5341 ctgaatagcg ccgtcgacca tcatcatcat catcattgac cggtcttgct agattctaat
     5401 caagaggatg tcagaatgcc atttgcctga gagatgcagg cttcattttt gatacttttt
     5461 tatttgtaac ctatatagta taggattttt tttgtcattt tgtttcttct cgtacgagct
     5521 tgctcctgat cagcctatct cgcagctgat gaatatcttg tggtaggggt ttgggaaaat
     5581 cattcgagtt tgatgttttt cttggtattt cccactcctc ttcagagtac agaagattaa
     5641 gtgagacctt cgtttgtgcg gatcccccac acaccatagc ttcaaaatgt ttctactcct
     5701 tttttactct tccagatttt ctcggactcc gcgcatcgcc gtaccacttc aaaacaccca
     5761 agcacagcat actaaatttt ccctctttct tcctctaggg tgtcgttaat tacccgtact
     5821 aaaggtttgg aaaagaaaaa agagaccgcc tcgtttcttt ttcttcgtcg aaaaaggcaa
     5881 taaaaatttt tatcacgttt ctttttcttg aaattttttt ttttagtttt tttctctttc
     5941 agtgacctcc attgatattt aagttaataa acggtcttca atttctcaag tttcagtttc
     6001 atttttcttg ttctattaca acttttttta cttcttgttc attagaaaga aagcatagca
     6061 atctaatcta aggggcggtg ttgacaatta atcatcggca tagtatatcg gcatagtata
     6121 atacgacaag gtgaggaact aaaccatggc caagttgacc agtgcc
//
