LOCUS       sequence-147680-        6161 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP. Figure 3. Strain 2..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6161)
  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 6161)
  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: PP296
FEATURES             Location/Qualifiers
     source          1..6161
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    343..392
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="attB site for the Mycopbacteriophage Bxb1 integrase 
                     (Xu et al., 2013)"
     CDS             1177..1197
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2416..2565
                     /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             2572..2598
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      2640..2886
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             join(3461..3463,3464..3466,3467..4177)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3461..3463,3464..3466,3467..4177)
                     /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(3506..3527)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3767..3786)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4114..4135
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4202..4231
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             4247..4264
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      4288..4534
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        4549..4960
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        4968..5015
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5034..5408
                     /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      5474..5721
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(5490..5508)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(join(5796..6161,1..223))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5876..5895)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
       61 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
      121 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
      181 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
      241 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
      301 atcagatctg agctcgtttg gccgtggccg tgctcgtcct cgtcggccgg cttgtcgacg
      361 acggcggtct ccgtcgtcag gatcatccgg gccacaagct tgctgacaga agcctcaaga
      421 aaaaaaaaat tcttcttcga ctatgctgga ggcagagatg atcgagccgg tagttaacta
      481 tatatagcta aattggttcc atcacctcga gtcagcatct ggttacgtaa ctctggcaac
      541 cagtaacacg cttaaggttt ggaacaacac taaactacct tgcggtacta ccattgacac
      601 tacacatcct taattccaat cctgtctggc ctccttcacc ttttaaccat cttgcccatt
      661 ccaactcgtg tcagattgcg tatcaagtga aaaaaaaaaa ttttaaaatc tttaacccaa
      721 tcaggtaata actgtcgcct cttttatctg ccgcactgca tgaggtgtcc ccttagtggg
      781 aaagagtact gagccaaccc tggaggacag caagggaaaa atacctacaa cttgcttcat
      841 aatggtcgta aaaacaatcc ttgtcggata taagtgttgt agactgtccc ttatcctctg
      901 cgatgttctt cctctcaaag tttgcgattt ctctctatca gaattgccat caagagactc
      961 aggactaatt tcgcagtccc acacgcactc gtacatgatt ggctgaaatt tccctaaaga
     1021 atttcttttt cacgaaaatt tttttttaca caagattttc agcagatata aaatggagag
     1081 caggacctcc gctgtgactc ttcttttttt tcttttattc tcactacata cattttagtt
     1141 attcgccaac ggcaactagt aaaaatgcgc ggagctccta agaaaaagcg caaagtccgg
     1201 ccggcatcta gacccgggga gcgccccttc cagtgtcgca tttgcatgcg gaacttttcg
     1261 cgccaggaca ggcttgacag gcatacccgt actcataccg gtgaaaaacc gtttcagtgt
     1321 cggatctgta tgcgaaattt ctcccagaag gagcacttgg cggggcatct acgtacgcac
     1381 accggcgaga agccattcca atgccgaata tgcatgcgca acttcagtcg ccgcgacaac
     1441 ctgaaccggc acctaaaaac ccacctgagg aacatatgcg gcggaggcac acctgcagct
     1501 gcgtcgactc tagaggatcc atctgctgga gacatgagag ctgccaacct ttggccaagc
     1561 ccgctcatga tcaaacgctc taagaagaac agcctggcct tgtccctgac ggccgaccag
     1621 atggtcagtg ccttgttgga tgctgagccc cccatactct attccgagta tgatcctacc
     1681 agacccttca gtgaagcttc gatgatgggc ttactgacca acctggcaga cagggagctg
     1741 gttcacatga tcaactgggc gaagagggtg ccaggctttg tggatttgac cctccatgat
     1801 caggtccacc ttctagaatg tgcctggcta gagatcctga tgattggtct cgtctggcgc
     1861 tccatggagc acccagtgaa gctactgttt gctcctaact tgctcttgga caggaaccag
     1921 ggaaaatgtg tagagggcat ggtggagatc ttcgacatgc tgctggctac atcatctcgg
     1981 ttccgcatga tgaatctgca gggagaggag tttgtgtgcc tcaaatctat tattttgctt
     2041 aattctggag tgtacacatt tctgtccagc accctgaagt ctctggaaga gaaggaccat
     2101 atccaccgag tcctggacaa gatcacagac actttgatcc acctgatggc caaggcaggc
     2161 ctgaccctgc agcagcagca ccagcggctg gcccagctcc tcctcatcct ctcccacatc
     2221 aggcacatga gtaacaaagg catggagcat ctgtacagca tgaagtgcaa gaacgtggtg
     2281 cccctctatg acctgctgct ggagatgctg gacgcccacc gcctacatgc gcccactagc
     2341 cgtggagggg catccgtgga ggagacggac caaagccact tggccactgc gggctctact
     2401 tcatcgccta gggccgacgc gctggacgat ttcgatctcg acatgctggg ttctgatgcc
     2461 ctcgatgact ttgacctgga tatgttggga agcgacgcat tggatgactt tgatctggac
     2521 atgctcggct ccgatgctct ggacgatttc gatctcgata tgttaattaa ctacccgtac
     2581 gacgttccgg actacgcttc ttgaggtacc atcggtagac cggtcttgct agattctaat
     2641 caagaggatg tcagaatgcc atttgcctga gagatgcagg cttcattttt gatacttttt
     2701 tatttgtaac ctatatagta taggattttt tttgtcattt tgtttcttct cgtacgagct
     2761 tgctcctgat cagcctatct cgcagctgat gaatatcttg tggtaggggt ttgggaaaat
     2821 cattcgagtt tgatgttttt cttggtattt cccactcctc ttcagagtac agaagattaa
     2881 gtgagagcta gcatatcata tagaagtcat cgcggcagat caattcatca atcggagtga
     2941 ggatcatcag tcagaatagg ggtattgtga atttactact attaatagaa gtgaggattt
     3001 acaattggtt actgttcaaa gttcaatcgg agttcgtatt caattgttgc tggaagctgc
     3061 tcaccagtca gagtaagggt gcaccggatg ggccgttccc ccatctatcg gtgtcagaat
     3121 ctgtgtgaat ggggccgaat ttatcaacag atccgaggac atagaagtta tcgagtccac
     3181 tcaccagtct gaataagggt acactcctcc aatcgatgtg acttcaattg gggtgagaat
     3241 cggaaccagg agagtgtcat accctatggc gcgccaccct atggcatgca tgtgctctgt
     3301 atgtatataa aactcttgtt ttcttctttt ctctaaatat tctttcctta tacattaggt
     3361 cctttgtagc ataaattact atacttctat agacacgcaa acacaaatac acacactaaa
     3421 ttaccggatc aattcgaagc ttaaatcgaa acgcgaattc atggtgagca agggcgagga
     3481 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     3541 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     3601 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     3661 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     3721 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     3781 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     3841 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     3901 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     3961 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     4021 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagagcgc
     4081 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     4141 cgccgggatc actctcggca tggacgagct gtacaaggcc gcggcggccg ccagctttct
     4201 agaacaaaaa ctcatctcag aagaggatct gaatagcgcc gtcgaccatc atcatcatca
     4261 tcattgaccg gtcttgctag attctaatca agaggatgtc agaatgccat ttgcctgaga
     4321 gatgcaggct tcatttttga tactttttta tttgtaacct atatagtata ggattttttt
     4381 tgtcattttg tttcttctcg tacgagcttg ctcctgatca gcctatctcg cagctgatga
     4441 atatcttgtg gtaggggttt gggaaaatca ttcgagtttg atgtttttct tggtatttcc
     4501 cactcctctt cagagtacag aagattaagt gagaccttcg tttgtgcgga tcccccacac
     4561 accatagctt caaaatgttt ctactccttt tttactcttc cagattttct cggactccgc
     4621 gcatcgccgt accacttcaa aacacccaag cacagcatac taaattttcc ctctttcttc
     4681 ctctagggtg tcgttaatta cccgtactaa aggtttggaa aagaaaaaag agaccgcctc
     4741 gtttcttttt cttcgtcgaa aaaggcaata aaaattttta tcacgtttct ttttcttgaa
     4801 attttttttt ttagtttttt tctctttcag tgacctccat tgatatttaa gttaataaac
     4861 ggtcttcaat ttctcaagtt tcagtttcat ttttcttgtt ctattacaac tttttttact
     4921 tcttgttcat tagaaagaaa gcatagcaat ctaatctaag gggcggtgtt gacaattaat
     4981 catcggcata gtatatcggc atagtataat acgacaaggt gaggaactaa accatggcca
     5041 agttgaccag tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct
     5101 ggaccgaccg gctcgggttc tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc
     5161 gggacgacgt gaccctgttc atcagcgcgg tccaggacca ggtggtgccg gacaacaccc
     5221 tggcctgggt gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt
     5281 ccacgaactt ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg
     5341 ggcgggagtt cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc
     5401 aggactgaca cgtccgacgg cggcccacgg gtcccaggcc tcggagatcc gtcccccttt
     5461 tcctttgtcg atatcatgta attagttatg tcacgcttac attcacgccc tccccccaca
     5521 tccgctctaa ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt
     5581 tttatagtta tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg
     5641 tacagacgcg tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga
     5701 cgctcgaagg ctttaatttg caagctggag accaacatgt gagcaaaagg ccagcaaaag
     5761 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     5821 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5881 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5941 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc
     6001 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     6061 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     6121 agacacgact tatcgccact ggcagcagcc actggtaaca g
//
