LOCUS       sequence-147725-        8601 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  246R. Promoter: GAP6, minimal Promoter: AOX1. Figure 5. Expresses 
            GFP and RFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8601)
  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 8601)
  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: PP311
FEATURES             Location/Qualifiers
     source          1..8601
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(19..607)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(99..118)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    727..776
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="attB site for the Mycopbacteriophage Bxb1 integrase 
                     (Xu et al., 2013)"
     protein_bind    1939..1957
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1960..1978
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             2048..2068
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3287..3436
                     /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             3443..3469
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      3511..3757
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             join(4336..4338,4339..4341,4342..5052)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4336..4338,4339..4341,4342..5052)
                     /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(4381..4402)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4642..4661)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4989..5010
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5077..5106
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             5122..5139
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      5163..5409
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        5424..5835
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        5843..5890
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5909..6283
                     /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      6349..6596
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(6365..6383)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     promoter        6611..7549
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     terminator      8313..8559
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
ORIGIN
        1 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
       61 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
      121 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
      181 ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc
      241 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
      301 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
      361 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
      421 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc
      481 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
      541 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
      601 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
      661 acgttaaggg attttggtca tgagatcaga tctgagctcg tttggccgtg gccgtgctcg
      721 tcctcgtcgg ccggcttgtc gacgacggcg gtctccgtcg tcaggatcat ccgggccaca
      781 agcttgctga cagaagcctc aagaaaaaaa aaattcttct tcgactatgc tggaggcaga
      841 gatgatcgag ccggtagtta actatatata gctaaattgg ttccatcacc tcgagtctat
      901 tcttgattta gtatacacat aaccaaattt ggatcaagtt tgaagtaaaa ctttaacttc
      961 agctccttac atttgcacta agatctctgc tactctggtc ccaagtgaac caccttttgg
     1021 accctattga ccggacctta acttgccaaa cctaaacgct taatgcctca gacgttttaa
     1081 tgcctctcaa cacctccaag gttgctttct tgagcatgcc tactagggac tttaacgaac
     1141 tgtggggttg cagacagttt caggcgtgtc ccgaccaata tggcctacta gactctctga
     1201 aaaatcacag ttttccagta gttccgatca aattaccatc gaaatggtcc cataaacgga
     1261 catttgacat ccgttcctga attatagtct tccaccgtgg atcatggtgt tccttttttt
     1321 cccaaagaat atcagcatcc cttaactacg ttaggtcagt gatgacaatg gaccaaattg
     1381 ttgcaaggtt tttctttttc tttcatcggc acatttcagc ctcacatgcg actattatcg
     1441 atcaatgaaa tccatcaaga ttgaaatctt aaaattgccc ctttcacttg acaggatcct
     1501 tttttgtaga aatgtcttgg tgtcctcgtc caatcaggta gccatctctg aaatatctgg
     1561 ctccgttgca actccgaacg acctgctggc aacgtaaaat tctccggggt aaaactttaa
     1621 tgtggagtaa tggaaccaga aacgtctctt cccttctctc tccttccacc gcccgttacc
     1681 gtccctagga aattttactc tgctggagag cttcttctac ggcccccttg cagcaatgct
     1741 cttcccagca ttacgttgcg ggtaaaacgg aggtcgtgta cccgacctag cagcccaggg
     1801 atggaaaagt cccggccgtc gctggcaata atagcgggcg gacgcatgtc atgagattat
     1861 tggaaaccac cagaatcgaa tataaaaggc gaacaccttt cccaattttg gtttctcctg
     1921 acccaaagac tttaaatttc cctatcagtg atagagatct ccctatcagt gatagagata
     1981 atttatttgt ccctatttca atcaattgaa caactatcaa aacacactag taaaaatgcg
     2041 cggagctcct aagaaaaagc gcaaagtccg gccggcatct agacccgggg agcgcccctt
     2101 ccagtgtcgc atttgcatgc ggaacttttc gcgccaggac aggcttgaca ggcatacccg
     2161 tactcatacc ggtgaaaaac cgtttcagtg tcggatctgt atgcgaaatt tctcccagaa
     2221 ggagcacttg gcggggcatc tacgtacgca caccggcgag aagccattcc aatgccgaat
     2281 atgcatgcgc aacttcagtc gccgcgacaa cctgaaccgg cacctaaaaa cccacctgag
     2341 gaacatatgc ggcggaggca cacctgcagc tgcgtcgact ctagaggatc catctgctgg
     2401 agacatgaga gctgccaacc tttggccaag cccgctcatg atcaaacgct ctaagaagaa
     2461 cagcctggcc ttgtccctga cggccgacca gatggtcagt gccttgttgg atgctgagcc
     2521 ccccatactc tattccgagt atgatcctac cagacccttc agtgaagctt cgatgatggg
     2581 cttactgacc aacctggcag acagggagct ggttcacatg atcaactggg cgaagagggt
     2641 gccaggcttt gtggatttga ccctccatga tcaggtccac cttctagaat gtgcctggct
     2701 agagatcctg atgattggtc tcgtctggcg ctccatggag cacccagtga agctactgtt
     2761 tgctcctaac ttgctcttgg acaggaacca gggaaaatgt gtagagggca tggtggagat
     2821 cttcgacatg ctgctggcta catcatctcg gttccgcatg atgaatctgc agggagagga
     2881 gtttgtgtgc ctcaaatcta ttattttgct taattctgga gtgtacacat ttctgtccag
     2941 caccctgaag tctctggaag agaaggacca tatccaccga gtcctggaca agatcacaga
     3001 cactttgatc cacctgatgg ccaaggcagg cctgaccctg cagcagcagc accagcggct
     3061 ggcccagctc ctcctcatcc tctcccacat caggcacatg agtaacaaag gcatggagca
     3121 tctgtacagc atgaagtgca agaacgtggt gcccctctat gacctgctgc tggagatgct
     3181 ggacgcccac cgcctacatg cgcccactag ccgtggaggg gcatccgtgg aggagacgga
     3241 ccaaagccac ttggccactg cgggctctac ttcatcgcct agggccgacg cgctggacga
     3301 tttcgatctc gacatgctgg gttctgatgc cctcgatgac tttgacctgg atatgttggg
     3361 aagcgacgca ttggatgact ttgatctgga catgctcggc tccgatgctc tggacgattt
     3421 cgatctcgat atgttaatta actacccgta cgacgttccg gactacgctt cttgaggtac
     3481 catcggtaga ccggtcttgc tagattctaa tcaagaggat gtcagaatgc catttgcctg
     3541 agagatgcag gcttcatttt tgatactttt ttatttgtaa cctatatagt ataggatttt
     3601 ttttgtcatt ttgtttcttc tcgtacgagc ttgctcctga tcagcctatc tcgcagctga
     3661 tgaatatctt gtggtagggg tttgggaaaa tcattcgagt ttgatgtttt tcttggtatt
     3721 tcccactcct cttcagagta cagaagatta agtgagagct agcatatcat atagaagtca
     3781 tcgcggcaga tcaattcatc aatcggagtg aggatattgc gagtttacca ccatcaatcg
     3841 gagtgaggat ctccaattgg tgacggtcca gtcatcaatc ggagtgagga ttcagctgct
     3901 tctcgaggcc gcacatcaat cggagtgagg atgtacaggg tgggctgttc caccatcaat
     3961 cggagtgagg atttgcgtca atggggcgga gttcatcaat cggagtgagg atatcgaagt
     4021 catcgagagc actcatcaat cggagtgagg atatactcca cccattgacg tcacatcaat
     4081 cggagtgagg attggaacca gaagggtctc ttcatcaatc ggagtgagga taccctatgg
     4141 gcgcgcctaa cccctacttg acagcaatat ataaacagaa ggaagctgcc ctgtcttaaa
     4201 cctttttttt tatcatcatt attagcttac tttcataatt gcgactggtt ccaattgaca
     4261 agcttttgat tttaacgact tttaacgaca acttgagaag atcaaaaaac aactaattat
     4321 tcgaaacgcg aattcatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     4381 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     4441 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     4501 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac
     4561 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     4621 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     4681 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     4741 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
     4801 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
     4861 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     4921 ctgcccgaca accactacct gagcacccag agcgccctga gcaaagaccc caacgagaag
     4981 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     5041 gagctgtaca aggccgcggc ggccgccagc tttctagaac aaaaactcat ctcagaagag
     5101 gatctgaata gcgccgtcga ccatcatcat catcatcatt gaccggtctt gctagattct
     5161 aatcaagagg atgtcagaat gccatttgcc tgagagatgc aggcttcatt tttgatactt
     5221 ttttatttgt aacctatata gtataggatt ttttttgtca ttttgtttct tctcgtacga
     5281 gcttgctcct gatcagccta tctcgcagct gatgaatatc ttgtggtagg ggtttgggaa
     5341 aatcattcga gtttgatgtt tttcttggta tttcccactc ctcttcagag tacagaagat
     5401 taagtgagac cttcgtttgt gcggatcccc cacacaccat agcttcaaaa tgtttctact
     5461 ccttttttac tcttccagat tttctcggac tccgcgcatc gccgtaccac ttcaaaacac
     5521 ccaagcacag catactaaat tttccctctt tcttcctcta gggtgtcgtt aattacccgt
     5581 actaaaggtt tggaaaagaa aaaagagacc gcctcgtttc tttttcttcg tcgaaaaagg
     5641 caataaaaat ttttatcacg tttctttttc ttgaaatttt tttttttagt ttttttctct
     5701 ttcagtgacc tccattgata tttaagttaa taaacggtct tcaatttctc aagtttcagt
     5761 ttcatttttc ttgttctatt acaacttttt ttacttcttg ttcattagaa agaaagcata
     5821 gcaatctaat ctaaggggcg gtgttgacaa ttaatcatcg gcatagtata tcggcatagt
     5881 ataatacgac aaggtgagga actaaaccat ggccaagttg accagtgccg ttccggtgct
     5941 caccgcgcgc gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttctcccg
     6001 ggacttcgtg gaggacgact tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag
     6061 cgcggtccag gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct
     6121 ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg
     6181 gccggccatg accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc
     6241 ggccggcaac tgcgtgcact tcgtggccga ggagcaggac tgacacgtcc gacggcggcc
     6301 cacgggtccc aggcctcgga gatccgtccc ccttttcctt tgtcgatatc atgtaattag
     6361 ttatgtcacg cttacattca cgccctcccc ccacatccgc tctaaccgaa aaggaaggag
     6421 ttagacaacc tgaagtctag gtccctattt atttttttat agttatgtta gtattaagaa
     6481 cgttatttat atttcaaatt tttctttttt ttctgtacag acgcgtgtac gcatgtaaca
     6541 ttatactgaa aaccttgctt gagaaggttt tgggacgctc gaaggcttta atttgcaagc
     6601 tggagaccaa gatctaacat ccaaagacga aaggttgaat gaaacctttt tgccatccga
     6661 catccacagg tccattctca cacataagtg ccaaacgcaa caggagggga tacactagca
     6721 gcagaccgtt gcaaacgcag gacctccact cctcttctcc tcaacaccca cttttgccat
     6781 cgaaaaacca gcccagttat tgggcttgat tggagctcgc tcattccaat tccttctatt
     6841 aggctactaa caccatgact ttattagcct gtctatcctg gcccccctgg cgaggttcat
     6901 gtttgtttat ttccgaatgc aacaagctcc gcattacacc cgaacatcac tccagatgag
     6961 ggctttctga gtgtggggtc aaatagtttc atgttcccca aatggcccaa aactgacagt
     7021 ttaaacgctg tcttggaacc taatatgaca aaagcgtgat ctcatccaag atgaactaag
     7081 tttggttcgt tgaaatgcta acggccagtt ggtcaaaaag aaacttccaa aagtcggcat
     7141 accgtttgtc ttgtttggta ttgattgacg aatgctcaaa aataatctca ttaatgctta
     7201 gcgcagtctc tctatcgctt ctgaaccccg gtgcacctgt gccgaaacgc aaatggggaa
     7261 acacccgctt tttggatgat tatgcattgt ctccacattg tatgcttcca agattctggt
     7321 gggaatactg ctgatagcct aacgttcatg atcaaaattt aactgttcta acccctactt
     7381 gacagcaata tataaacaga aggaagctgc cctgtcttaa accttttttt ttatcatcat
     7441 tattagctta ctttcataat tgcgactggt tccaattgac aagcttttga ttttaacgac
     7501 ttttaacgac aacttgagaa gatcaaaaaa caactaatta ttcgaaacga attcatggca
     7561 acttccggta tggtctcaaa gggagaggaa aataatatgg ctattattaa ggagtttatg
     7621 cgttttaagg tacatatgga aggttctgtc aacggtcacg aattcgaaat tgaaggtgag
     7681 ggggagggga gaccatacga gggaactcag actgctaagt taaaggtcac taaaggtggt
     7741 cctttacctt tcgcctggga tatcctgtct ccacagttta tgtacggttc aaaggcttat
     7801 gtgaaacatc ctgccgatat cccagattat cttaaacttt ctttccctga gggttttaag
     7861 tgggagaggg taatgaactt tgaagacggt ggtgtggtca ctgttactca ggactcaagt
     7921 ctgcaggacg gtgagttcat ctacaaggtg aagctgagag gtaccaattt tccatcagat
     7981 ggtcccgtga tgcaaaaaaa gacaatgggt tgggaagctt ctagtgaacg tatgtatccc
     8041 gaagatggag ctttgaaagg tgaaattaag caaagactaa aacttaagga tggtggacat
     8101 tacgatgctg aagttaagac gacctacaag gccaaaaagc cagtccagtt gcctggagca
     8161 tacaatgtta acatcaaatt ggatataact tcccataatg aagactatac catcgtcgag
     8221 caatacgaac gagccgaagg gagacacagt actggtggta tggatgaact ttataaagga
     8281 tccggaaccg caccggtctt gctagattct aatcaagagg atgtcagaat gccatttgcc
     8341 tgagagatgc aggcttcatt tttgatactt ttttatttgt aacctatata gtataggatt
     8401 ttttttgtca ttttgtttct tctcgtacga gcttgctcct gatcagccta tctcgcagct
     8461 gatgaatatc ttgtggtagg ggtttgggaa aatcattcga gtttgatgtt tttcttggta
     8521 tttcccactc ctcttcagag tacagaagat taagtgagac atgtgagcaa aaggccagca
     8581 aaaggccagg aaccgtaaaa a
//
