LOCUS       sequence-147712-        5931 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Promoter: scTEF1, minimal Promoter: GCW14. Figure 4. Expresses GFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5931)
  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 5931)
  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: PP277
FEATURES             Location/Qualifiers
     source          1..5931
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      304..551
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(320..338)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(626..1214)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(706..725)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    1334..1383
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="attB site for the Mycopbacteriophage Bxb1 integrase 
                     (Xu et al., 2013)"
     promoter        1504..1910
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     CDS             1934..1954
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3173..3322
                     /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             3329..3355
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      3397..3643
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             join(4222..4224,4225..4227,4228..4938)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4222..4224,4225..4227,4228..4938)
                     /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(4267..4288)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4528..4547)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4875..4896
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4963..4992
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             5008..5025
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      5049..5295
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        5310..5721
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        5729..5776
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             join(5795..5931,1..238)
                     /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 gaccctgttc atcagcgcgg tccaggacca ggtggtgccg gacaacaccc tggcctgggt
       61 gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt
      121 ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg ggcgggagtt
      181 cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc aggactgaca
      241 cgtccgacgg cggcccacgg gtcccaggcc tcggagatcc gtcccccttt tcctttgtcg
      301 atatcatgta attagttatg tcacgcttac attcacgccc tccccccaca tccgctctaa
      361 ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt tttatagtta
      421 tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg tacagacgcg
      481 tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg
      541 ctttaatttg caagctggag accaacatgt gagcaaaagg ccagcaaaag gccaggaacc
      601 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
      661 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
      721 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
      781 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc
      841 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
      901 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
      961 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     1021 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     1081 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     1141 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     1201 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     1261 aaaactcacg ttaagggatt ttggtcatga gatcagatct gagctcgttt ggccgtggcc
     1321 gtgctcgtcc tcgtcggccg gcttgtcgac gacggcggtc tccgtcgtca ggatcatccg
     1381 ggccacaagc ttgctgacag aagcctcaag aaaaaaaaaa ttcttcttcg actatgctgg
     1441 aggcagagat gatcgagccg gtagttaact atatatagct aaattggttc catcacctcg
     1501 agtccacaca ccatagcttc aaaatgtttc tactcctttt ttactcttcc agattttctc
     1561 ggactccgcg catcgccgta ccacttcaaa acacccaagc acagcatact aaatttcccc
     1621 tctttcttcc tctagggtgt cgttaattac ccgtactaaa ggtttggaaa agaaaaaaga
     1681 gaccgcctcg tttctttttc ttcgtcgaaa aaggcaataa aaatttttat cacgtttctt
     1741 tttcttgaaa attttttttt tgattttttt ctctttcgat gacctcccat tgatatttaa
     1801 gttaataaac ggtcttcaat ttctcaagtt tcagtttcat ttttcttgtt ctattacaac
     1861 tttttttact tcttgctcat tagaaagaaa gcatagcaat ctaatctaag gactagtaaa
     1921 aatgcgcgga gctcctaaga aaaagcgcaa agtccggccg gcatctagac ccggggagcg
     1981 ccccttccag tgtcgcattt gcatgcggaa cttttcgcgc caggacaggc ttgacaggca
     2041 tacccgtact cataccggtg aaaaaccgtt tcagtgtcgg atctgtatgc gaaatttctc
     2101 ccagaaggag cacttggcgg ggcatctacg tacgcacacc ggcgagaagc cattccaatg
     2161 ccgaatatgc atgcgcaact tcagtcgccg cgacaacctg aaccggcacc taaaaaccca
     2221 cctgaggaac atatgcggcg gaggcacacc tgcagctgcg tcgactctag aggatccatc
     2281 tgctggagac atgagagctg ccaacctttg gccaagcccg ctcatgatca aacgctctaa
     2341 gaagaacagc ctggccttgt ccctgacggc cgaccagatg gtcagtgcct tgttggatgc
     2401 tgagcccccc atactctatt ccgagtatga tcctaccaga cccttcagtg aagcttcgat
     2461 gatgggctta ctgaccaacc tggcagacag ggagctggtt cacatgatca actgggcgaa
     2521 gagggtgcca ggctttgtgg atttgaccct ccatgatcag gtccaccttc tagaatgtgc
     2581 ctggctagag atcctgatga ttggtctcgt ctggcgctcc atggagcacc cagtgaagct
     2641 actgtttgct cctaacttgc tcttggacag gaaccaggga aaatgtgtag agggcatggt
     2701 ggagatcttc gacatgctgc tggctacatc atctcggttc cgcatgatga atctgcaggg
     2761 agaggagttt gtgtgcctca aatctattat tttgcttaat tctggagtgt acacatttct
     2821 gtccagcacc ctgaagtctc tggaagagaa ggaccatatc caccgagtcc tggacaagat
     2881 cacagacact ttgatccacc tgatggccaa ggcaggcctg accctgcagc agcagcacca
     2941 gcggctggcc cagctcctcc tcatcctctc ccacatcagg cacatgagta acaaaggcat
     3001 ggagcatctg tacagcatga agtgcaagaa cgtggtgccc ctctatgacc tgctgctgga
     3061 gatgctggac gcccaccgcc tacatgcgcc cactagccgt ggaggggcat ccgtggagga
     3121 gacggaccaa agccacttgg ccactgcggg ctctacttca tcgcctaggg ccgacgcgct
     3181 ggacgatttc gatctcgaca tgctgggttc tgatgccctc gatgactttg acctggatat
     3241 gttgggaagc gacgcattgg atgactttga tctggacatg ctcggctccg atgctctgga
     3301 cgatttcgat ctcgatatgt taattaacta cccgtacgac gttccggact acgcttcttg
     3361 aggtaccatc ggtagaccgg tcttgctaga ttctaatcaa gaggatgtca gaatgccatt
     3421 tgcctgagag atgcaggctt catttttgat acttttttat ttgtaaccta tatagtatag
     3481 gatttttttt gtcattttgt ttcttctcgt acgagcttgc tcctgatcag cctatctcgc
     3541 agctgatgaa tatcttgtgg taggggtttg ggaaaatcat tcgagtttga tgtttttctt
     3601 ggtatttccc actcctcttc agagtacaga agattaagtg agagctagca tatcatatag
     3661 aagtcatcgc ggcagatcaa ttcatcaatc ggagtgagga tattgcgagt ttaccaccat
     3721 caatcggagt gaggatctcc aattggtgac ggtccagtca tcaatcggag tgaggattca
     3781 gctgcttctc gaggccgcac atcaatcgga gtgaggatgt acagggtggg ctgttccacc
     3841 atcaatcgga gtgaggattt gcgtcaatgg ggcggagttc atcaatcgga gtgaggatat
     3901 cgaagtcatc gagagcactc atcaatcgga gtgaggatat actccaccca ttgacgtcac
     3961 atcaatcgga gtgaggattg gaaccagaag ggtctcttca tcaatcggag tgaggatacc
     4021 ctatgggcgc gcctagggca gattttgagt ttaaaatagg aaatataaac aaatataccg
     4081 cgaaaaaggt ttgtttatag cttttcgcct ggtgccgtac ggtataaata catactctcc
     4141 tcccccccct ggttctcttt ttcttttgtt acttacattt taccgttccg tcactcgctt
     4201 cactcaacaa caaaagaatt catggtgagc aagggcgagg agctgttcac cggggtggtg
     4261 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
     4321 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
     4381 ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc
     4441 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
     4501 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
     4561 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     4621 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc
     4681 atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag
     4741 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
     4801 gtgctgctgc ccgacaacca ctacctgagc acccagagcg ccctgagcaa agaccccaac
     4861 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
     4921 atggacgagc tgtacaaggc cgcggcggcc gccagctttc tagaacaaaa actcatctca
     4981 gaagaggatc tgaatagcgc cgtcgaccat catcatcatc atcattgacc ggtcttgcta
     5041 gattctaatc aagaggatgt cagaatgcca tttgcctgag agatgcaggc ttcatttttg
     5101 atactttttt atttgtaacc tatatagtat aggatttttt ttgtcatttt gtttcttctc
     5161 gtacgagctt gctcctgatc agcctatctc gcagctgatg aatatcttgt ggtaggggtt
     5221 tgggaaaatc attcgagttt gatgtttttc ttggtatttc ccactcctct tcagagtaca
     5281 gaagattaag tgagaccttc gtttgtgcgg atcccccaca caccatagct tcaaaatgtt
     5341 tctactcctt ttttactctt ccagattttc tcggactccg cgcatcgccg taccacttca
     5401 aaacacccaa gcacagcata ctaaattttc cctctttctt cctctagggt gtcgttaatt
     5461 acccgtacta aaggtttgga aaagaaaaaa gagaccgcct cgtttctttt tcttcgtcga
     5521 aaaaggcaat aaaaattttt atcacgtttc tttttcttga aatttttttt tttagttttt
     5581 ttctctttca gtgacctcca ttgatattta agttaataaa cggtcttcaa tttctcaagt
     5641 ttcagtttca tttttcttgt tctattacaa ctttttttac ttcttgttca ttagaaagaa
     5701 agcatagcaa tctaatctaa ggggcggtgt tgacaattaa tcatcggcat agtatatcgg
     5761 catagtataa tacgacaagg tgaggaacta aaccatggcc aagttgacca gtgccgttcc
     5821 ggtgctcacc gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt
     5881 ctcccgggac ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg t
//
