LOCUS       sequence-147709-        5931 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Promoter: scTEF1, minimal Promoter: AOX1. 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: PP247
FEATURES             Location/Qualifiers
     source          1..5931
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(46..634)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(126..145)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    754..803
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="attB site for the Mycopbacteriophage Bxb1 integrase 
                     (Xu et al., 2013)"
     promoter        924..1330
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     CDS             1354..1374
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2593..2742
                     /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             2749..2775
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      2817..3063
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             join(3642..3644,3645..3647,3648..4358)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3642..3644,3645..3647,3648..4358)
                     /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(3687..3708)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3948..3967)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4295..4316
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4383..4412
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             4428..4445
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      4469..4715
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        4730..5141
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        5149..5196
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5215..5589
                     /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      5655..5902
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(5671..5689)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
ORIGIN
        1 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
       61 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
      121 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
      181 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
      241 atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
      301 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
      361 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
      421 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
      481 tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
      541 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
      601 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
      661 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gatcagatct
      721 gagctcgttt ggccgtggcc gtgctcgtcc tcgtcggccg gcttgtcgac gacggcggtc
      781 tccgtcgtca ggatcatccg ggccacaagc ttgctgacag aagcctcaag aaaaaaaaaa
      841 ttcttcttcg actatgctgg aggcagagat gatcgagccg gtagttaact atatatagct
      901 aaattggttc catcacctcg agtccacaca ccatagcttc aaaatgtttc tactcctttt
      961 ttactcttcc agattttctc ggactccgcg catcgccgta ccacttcaaa acacccaagc
     1021 acagcatact aaatttcccc tctttcttcc tctagggtgt cgttaattac ccgtactaaa
     1081 ggtttggaaa agaaaaaaga gaccgcctcg tttctttttc ttcgtcgaaa aaggcaataa
     1141 aaatttttat cacgtttctt tttcttgaaa attttttttt tgattttttt ctctttcgat
     1201 gacctcccat tgatatttaa gttaataaac ggtcttcaat ttctcaagtt tcagtttcat
     1261 ttttcttgtt ctattacaac tttttttact tcttgctcat tagaaagaaa gcatagcaat
     1321 ctaatctaag gactagtaaa aatgcgcgga gctcctaaga aaaagcgcaa agtccggccg
     1381 gcatctagac ccggggagcg ccccttccag tgtcgcattt gcatgcggaa cttttcgcgc
     1441 caggacaggc ttgacaggca tacccgtact cataccggtg aaaaaccgtt tcagtgtcgg
     1501 atctgtatgc gaaatttctc ccagaaggag cacttggcgg ggcatctacg tacgcacacc
     1561 ggcgagaagc cattccaatg ccgaatatgc atgcgcaact tcagtcgccg cgacaacctg
     1621 aaccggcacc taaaaaccca cctgaggaac atatgcggcg gaggcacacc tgcagctgcg
     1681 tcgactctag aggatccatc tgctggagac atgagagctg ccaacctttg gccaagcccg
     1741 ctcatgatca aacgctctaa gaagaacagc ctggccttgt ccctgacggc cgaccagatg
     1801 gtcagtgcct tgttggatgc tgagcccccc atactctatt ccgagtatga tcctaccaga
     1861 cccttcagtg aagcttcgat gatgggctta ctgaccaacc tggcagacag ggagctggtt
     1921 cacatgatca actgggcgaa gagggtgcca ggctttgtgg atttgaccct ccatgatcag
     1981 gtccaccttc tagaatgtgc ctggctagag atcctgatga ttggtctcgt ctggcgctcc
     2041 atggagcacc cagtgaagct actgtttgct cctaacttgc tcttggacag gaaccaggga
     2101 aaatgtgtag agggcatggt ggagatcttc gacatgctgc tggctacatc atctcggttc
     2161 cgcatgatga atctgcaggg agaggagttt gtgtgcctca aatctattat tttgcttaat
     2221 tctggagtgt acacatttct gtccagcacc ctgaagtctc tggaagagaa ggaccatatc
     2281 caccgagtcc tggacaagat cacagacact ttgatccacc tgatggccaa ggcaggcctg
     2341 accctgcagc agcagcacca gcggctggcc cagctcctcc tcatcctctc ccacatcagg
     2401 cacatgagta acaaaggcat ggagcatctg tacagcatga agtgcaagaa cgtggtgccc
     2461 ctctatgacc tgctgctgga gatgctggac gcccaccgcc tacatgcgcc cactagccgt
     2521 ggaggggcat ccgtggagga gacggaccaa agccacttgg ccactgcggg ctctacttca
     2581 tcgcctaggg ccgacgcgct ggacgatttc gatctcgaca tgctgggttc tgatgccctc
     2641 gatgactttg acctggatat gttgggaagc gacgcattgg atgactttga tctggacatg
     2701 ctcggctccg atgctctgga cgatttcgat ctcgatatgt taattaacta cccgtacgac
     2761 gttccggact acgcttcttg aggtaccatc ggtagaccgg tcttgctaga ttctaatcaa
     2821 gaggatgtca gaatgccatt tgcctgagag atgcaggctt catttttgat acttttttat
     2881 ttgtaaccta tatagtatag gatttttttt gtcattttgt ttcttctcgt acgagcttgc
     2941 tcctgatcag cctatctcgc agctgatgaa tatcttgtgg taggggtttg ggaaaatcat
     3001 tcgagtttga tgtttttctt ggtatttccc actcctcttc agagtacaga agattaagtg
     3061 agagctagca tatcatatag aagtcatcgc ggcagatcaa ttcatcaatc ggagtgagga
     3121 tattgcgagt ttaccaccat caatcggagt gaggatctcc aattggtgac ggtccagtca
     3181 tcaatcggag tgaggattca gctgcttctc gaggccgcac atcaatcgga gtgaggatgt
     3241 acagggtggg ctgttccacc atcaatcgga gtgaggattt gcgtcaatgg ggcggagttc
     3301 atcaatcgga gtgaggatat cgaagtcatc gagagcactc atcaatcgga gtgaggatat
     3361 actccaccca ttgacgtcac atcaatcgga gtgaggattg gaaccagaag ggtctcttca
     3421 tcaatcggag tgaggatacc ctatgggcgc gcctaacccc tacttgacag caatatataa
     3481 acagaaggaa gctgccctgt cttaaacctt tttttttatc atcattatta gcttactttc
     3541 ataattgcga ctggttccaa ttgacaagct tttgatttta acgactttta acgacaactt
     3601 gagaagatca aaaaacaact aattattcga aacgcgaatt catggtgagc aagggcgagg
     3661 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     3721 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     3781 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     3841 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     3901 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     3961 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     4021 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     4081 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     4141 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     4201 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagagcg
     4261 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     4321 ccgccgggat cactctcggc atggacgagc tgtacaaggc cgcggcggcc gccagctttc
     4381 tagaacaaaa actcatctca gaagaggatc tgaatagcgc cgtcgaccat catcatcatc
     4441 atcattgacc ggtcttgcta gattctaatc aagaggatgt cagaatgcca tttgcctgag
     4501 agatgcaggc ttcatttttg atactttttt atttgtaacc tatatagtat aggatttttt
     4561 ttgtcatttt gtttcttctc gtacgagctt gctcctgatc agcctatctc gcagctgatg
     4621 aatatcttgt ggtaggggtt tgggaaaatc attcgagttt gatgtttttc ttggtatttc
     4681 ccactcctct tcagagtaca gaagattaag tgagaccttc gtttgtgcgg atcccccaca
     4741 caccatagct tcaaaatgtt tctactcctt ttttactctt ccagattttc tcggactccg
     4801 cgcatcgccg taccacttca aaacacccaa gcacagcata ctaaattttc cctctttctt
     4861 cctctagggt gtcgttaatt acccgtacta aaggtttgga aaagaaaaaa gagaccgcct
     4921 cgtttctttt tcttcgtcga aaaaggcaat aaaaattttt atcacgtttc tttttcttga
     4981 aatttttttt tttagttttt ttctctttca gtgacctcca ttgatattta agttaataaa
     5041 cggtcttcaa tttctcaagt ttcagtttca tttttcttgt tctattacaa ctttttttac
     5101 ttcttgttca ttagaaagaa agcatagcaa tctaatctaa ggggcggtgt tgacaattaa
     5161 tcatcggcat agtatatcgg catagtataa tacgacaagg tgaggaacta aaccatggcc
     5221 aagttgacca gtgccgttcc ggtgctcacc gcgcgcgacg tcgccggagc ggtcgagttc
     5281 tggaccgacc ggctcgggtt ctcccgggac ttcgtggagg acgacttcgc cggtgtggtc
     5341 cgggacgacg tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc ggacaacacc
     5401 ctggcctggg tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc ggaggtcgtg
     5461 tccacgaact tccgggacgc ctccgggccg gccatgaccg agatcggcga gcagccgtgg
     5521 gggcgggagt tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt ggccgaggag
     5581 caggactgac acgtccgacg gcggcccacg ggtcccaggc ctcggagatc cgtccccctt
     5641 ttcctttgtc gatatcatgt aattagttat gtcacgctta cattcacgcc ctccccccac
     5701 atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt
     5761 ttttatagtt atgttagtat taagaacgtt atttatattt caaatttttc ttttttttct
     5821 gtacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga aggttttggg
     5881 acgctcgaag gctttaattt gcaagctgga gaccaacatg tgagcaaaag g
//
