LOCUS       sequence-147681-        6162 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP. Figure 3. Strain 3..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6162)
  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 6162)
  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: PP297
FEATURES             Location/Qualifiers
     source          1..6162
                     /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             1178..1198
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2417..2566
                     /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             2573..2599
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      2641..2887
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             join(3462..3464,3465..3467,3468..4178)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3462..3464,3465..3467,3468..4178)
                     /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(3507..3528)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3768..3787)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4115..4136
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4203..4232
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             4248..4265
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      4289..4535
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        4550..4961
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        4969..5016
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5035..5409
                     /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      5475..5722
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(5491..5509)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(join(5797..6162,1..223))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5877..5896)
                     /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 attttaaaat ctttaaccca
      721 atcaggtaat aactgtcgcc tcttttatct gccgcactgc atgaggtgtc cccttagtgg
      781 gaaagagtac tgagccaacc ctggaggaca gcaagggaaa aatacctaca acttgcttca
      841 taatggtcgt aaaaacaatc cttgtcggat ataagtgttg tagactgtcc cttatcctct
      901 gcgatgttct tcctctcaaa gtttgcgatt tctctctatc agaattgcca tcaagagact
      961 caggactaat ttcgcagtcc cacacgcact cgtacatgat tggctgaaat ttccctaaag
     1021 aatttctttt tcacgaaaat ttttttttac acaagatttt cagcagatat aaaatggaga
     1081 gcaggacctc cgctgtgact cttctttttt ttcttttatt ctcactacat acattttagt
     1141 tattcgccaa cggcaactag taaaaatgcg cggagctcct aagaaaaagc gcaaagtccg
     1201 gccggcatct agacccgggg agcgcccctt ccagtgtcgc atttgcatgc ggaacttttc
     1261 gcgccaggac aggcttgaca ggcatacccg tactcatacc ggtgaaaaac cgtttcagtg
     1321 tcggatctgt atgcgaaatt tctcccagaa ggagcacttg gcggggcatc tacgtacgca
     1381 caccggcgag aagccattcc aatgccgaat atgcatgcgc aacttcagtc gccgcgacaa
     1441 cctgaaccgg cacctaaaaa cccacctgag gaacatatgc ggcggaggca cacctgcagc
     1501 tgcgtcgact ctagaggatc catctgctgg agacatgaga gctgccaacc tttggccaag
     1561 cccgctcatg atcaaacgct ctaagaagaa cagcctggcc ttgtccctga cggccgacca
     1621 gatggtcagt gccttgttgg atgctgagcc ccccatactc tattccgagt atgatcctac
     1681 cagacccttc agtgaagctt cgatgatggg cttactgacc aacctggcag acagggagct
     1741 ggttcacatg atcaactggg cgaagagggt gccaggcttt gtggatttga ccctccatga
     1801 tcaggtccac cttctagaat gtgcctggct agagatcctg atgattggtc tcgtctggcg
     1861 ctccatggag cacccagtga agctactgtt tgctcctaac ttgctcttgg acaggaacca
     1921 gggaaaatgt gtagagggca tggtggagat cttcgacatg ctgctggcta catcatctcg
     1981 gttccgcatg atgaatctgc agggagagga gtttgtgtgc ctcaaatcta ttattttgct
     2041 taattctgga gtgtacacat ttctgtccag caccctgaag tctctggaag agaaggacca
     2101 tatccaccga gtcctggaca agatcacaga cactttgatc cacctgatgg ccaaggcagg
     2161 cctgaccctg cagcagcagc accagcggct ggcccagctc ctcctcatcc tctcccacat
     2221 caggcacatg agtaacaaag gcatggagca tctgtacagc atgaagtgca agaacgtggt
     2281 gcccctctat gacctgctgc tggagatgct ggacgcccac cgcctacatg cgcccactag
     2341 ccgtggaggg gcatccgtgg aggagacgga ccaaagccac ttggccactg cgggctctac
     2401 ttcatcgcct agggccgacg cgctggacga tttcgatctc gacatgctgg gttctgatgc
     2461 cctcgatgac tttgacctgg atatgttggg aagcgacgca ttggatgact ttgatctgga
     2521 catgctcggc tccgatgctc tggacgattt cgatctcgat atgttaatta actacccgta
     2581 cgacgttccg gactacgctt cttgaggtac catcggtaga ccggtcttgc tagattctaa
     2641 tcaagaggat gtcagaatgc catttgcctg agagatgcag gcttcatttt tgatactttt
     2701 ttatttgtaa cctatatagt ataggatttt ttttgtcatt ttgtttcttc tcgtacgagc
     2761 ttgctcctga tcagcctatc tcgcagctga tgaatatctt gtggtagggg tttgggaaaa
     2821 tcattcgagt ttgatgtttt tcttggtatt tcccactcct cttcagagta cagaagatta
     2881 agtgagagct agcatatcat atagaagtca tcgcggcaga tcaattcatc agtcagaata
     2941 ggggtattgt gaatttacta ctattaatag aagtgaggat ttacaattgg ttactgttca
     3001 aagttcaatc ggagttcgta ttcaattgtt gctggaagct gctcaccagt cagagtaagg
     3061 gtgcaccgga tgggccgttc ccccatcaat cggagtgagg atcatctatc ggtgtcagaa
     3121 tctgtgtgaa tggggccgaa tttatcaaca gatccgagga catagaagtt atcgagtcca
     3181 ctcaccagtc tgaataaggg tacactcctc caatcgatgt gacttcaatt ggggtgagaa
     3241 tcggaaccag gagagtgtca taccctatgg cgcgccaccc tatggcatgc atgtgctctg
     3301 tatgtatata aaactcttgt tttcttcttt tctctaaata ttctttcctt atacattagg
     3361 tcctttgtag cataaattac tatacttcta tagacacgca aacacaaata cacacactaa
     3421 attaccggat caattcgaag cttaaatcga aacgcgaatt catggtgagc aagggcgagg
     3481 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     3541 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     3601 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     3661 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     3721 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     3781 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     3841 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     3901 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     3961 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     4021 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagagcg
     4081 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     4141 ccgccgggat cactctcggc atggacgagc tgtacaaggc cgcggcggcc gccagctttc
     4201 tagaacaaaa actcatctca gaagaggatc tgaatagcgc cgtcgaccat catcatcatc
     4261 atcattgacc ggtcttgcta gattctaatc aagaggatgt cagaatgcca tttgcctgag
     4321 agatgcaggc ttcatttttg atactttttt atttgtaacc tatatagtat aggatttttt
     4381 ttgtcatttt gtttcttctc gtacgagctt gctcctgatc agcctatctc gcagctgatg
     4441 aatatcttgt ggtaggggtt tgggaaaatc attcgagttt gatgtttttc ttggtatttc
     4501 ccactcctct tcagagtaca gaagattaag tgagaccttc gtttgtgcgg atcccccaca
     4561 caccatagct tcaaaatgtt tctactcctt ttttactctt ccagattttc tcggactccg
     4621 cgcatcgccg taccacttca aaacacccaa gcacagcata ctaaattttc cctctttctt
     4681 cctctagggt gtcgttaatt acccgtacta aaggtttgga aaagaaaaaa gagaccgcct
     4741 cgtttctttt tcttcgtcga aaaaggcaat aaaaattttt atcacgtttc tttttcttga
     4801 aatttttttt tttagttttt ttctctttca gtgacctcca ttgatattta agttaataaa
     4861 cggtcttcaa tttctcaagt ttcagtttca tttttcttgt tctattacaa ctttttttac
     4921 ttcttgttca ttagaaagaa agcatagcaa tctaatctaa ggggcggtgt tgacaattaa
     4981 tcatcggcat agtatatcgg catagtataa tacgacaagg tgaggaacta aaccatggcc
     5041 aagttgacca gtgccgttcc ggtgctcacc gcgcgcgacg tcgccggagc ggtcgagttc
     5101 tggaccgacc ggctcgggtt ctcccgggac ttcgtggagg acgacttcgc cggtgtggtc
     5161 cgggacgacg tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc ggacaacacc
     5221 ctggcctggg tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc ggaggtcgtg
     5281 tccacgaact tccgggacgc ctccgggccg gccatgaccg agatcggcga gcagccgtgg
     5341 gggcgggagt tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt ggccgaggag
     5401 caggactgac acgtccgacg gcggcccacg ggtcccaggc ctcggagatc cgtccccctt
     5461 ttcctttgtc gatatcatgt aattagttat gtcacgctta cattcacgcc ctccccccac
     5521 atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt
     5581 ttttatagtt atgttagtat taagaacgtt atttatattt caaatttttc ttttttttct
     5641 gtacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga aggttttggg
     5701 acgctcgaag gctttaattt gcaagctgga gaccaacatg tgagcaaaag gccagcaaaa
     5761 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     5821 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     5881 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     5941 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg
     6001 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     6061 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     6121 aagacacgac ttatcgccac tggcagcagc cactggtaac ag
//
