LOCUS       sequence-147724-        8561 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  245R. Promoter: GAP, 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 8561)
  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 8561)
  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: PP310
FEATURES             Location/Qualifiers
     source          1..8561
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      190..437
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(206..224)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     promoter        452..1390
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     terminator      2154..2400
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     rep_origin      complement(2461..3049)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2541..2560)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    3169..3218
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="attB site for the Mycopbacteriophage Bxb1 integrase 
                     (Xu et al., 2013)"
     promoter        3943..4419
                     /label=GAP promoter
                     /note="promoter for Pichia pastoris 
                     glyceraldehyde-3-phosphate dehydrogenase"
     CDS             4450..4470
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5689..5838
                     /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             5845..5871
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      5913..6159
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             join(6738..6740,6741..6743,6744..7454)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(6738..6740,6741..6743,6744..7454)
                     /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(6783..6804)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(7044..7063)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7391..7412
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             7479..7508
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             7524..7541
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      7565..7811
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        7826..8237
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        8245..8292
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             join(8311..8561,1..124)
                     /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 gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc cgtgggggcg
       61 ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga
      121 ctgacacgtc cgacggcggc ccacgggtcc caggcctcgg agatccgtcc cccttttcct
      181 ttgtcgatat catgtaatta gttatgtcac gcttacattc acgccctccc cccacatccg
      241 ctctaaccga aaaggaagga gttagacaac ctgaagtcta ggtccctatt tattttttta
      301 tagttatgtt agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca
      361 gacgcgtgta cgcatgtaac attatactga aaaccttgct tgagaaggtt ttgggacgct
      421 cgaaggcttt aatttgcaag ctggagacca agatctaaca tccaaagacg aaaggttgaa
      481 tgaaaccttt ttgccatccg acatccacag gtccattctc acacataagt gccaaacgca
      541 acaggagggg atacactagc agcagaccgt tgcaaacgca ggacctccac tcctcttctc
      601 ctcaacaccc acttttgcca tcgaaaaacc agcccagtta ttgggcttga ttggagctcg
      661 ctcattccaa ttccttctat taggctacta acaccatgac tttattagcc tgtctatcct
      721 ggcccccctg gcgaggttca tgtttgttta tttccgaatg caacaagctc cgcattacac
      781 ccgaacatca ctccagatga gggctttctg agtgtggggt caaatagttt catgttcccc
      841 aaatggccca aaactgacag tttaaacgct gtcttggaac ctaatatgac aaaagcgtga
      901 tctcatccaa gatgaactaa gtttggttcg ttgaaatgct aacggccagt tggtcaaaaa
      961 gaaacttcca aaagtcggca taccgtttgt cttgtttggt attgattgac gaatgctcaa
     1021 aaataatctc attaatgctt agcgcagtct ctctatcgct tctgaacccc ggtgcacctg
     1081 tgccgaaacg caaatgggga aacacccgct ttttggatga ttatgcattg tctccacatt
     1141 gtatgcttcc aagattctgg tgggaatact gctgatagcc taacgttcat gatcaaaatt
     1201 taactgttct aacccctact tgacagcaat atataaacag aaggaagctg ccctgtctta
     1261 aacctttttt tttatcatca ttattagctt actttcataa ttgcgactgg ttccaattga
     1321 caagcttttg attttaacga cttttaacga caacttgaga agatcaaaaa acaactaatt
     1381 attcgaaacg aattcatggc aacttccggt atggtctcaa agggagagga aaataatatg
     1441 gctattatta aggagtttat gcgttttaag gtacatatgg aaggttctgt caacggtcac
     1501 gaattcgaaa ttgaaggtga gggggagggg agaccatacg agggaactca gactgctaag
     1561 ttaaaggtca ctaaaggtgg tcctttacct ttcgcctggg atatcctgtc tccacagttt
     1621 atgtacggtt caaaggctta tgtgaaacat cctgccgata tcccagatta tcttaaactt
     1681 tctttccctg agggttttaa gtgggagagg gtaatgaact ttgaagacgg tggtgtggtc
     1741 actgttactc aggactcaag tctgcaggac ggtgagttca tctacaaggt gaagctgaga
     1801 ggtaccaatt ttccatcaga tggtcccgtg atgcaaaaaa agacaatggg ttgggaagct
     1861 tctagtgaac gtatgtatcc cgaagatgga gctttgaaag gtgaaattaa gcaaagacta
     1921 aaacttaagg atggtggaca ttacgatgct gaagttaaga cgacctacaa ggccaaaaag
     1981 ccagtccagt tgcctggagc atacaatgtt aacatcaaat tggatataac ttcccataat
     2041 gaagactata ccatcgtcga gcaatacgaa cgagccgaag ggagacacag tactggtggt
     2101 atggatgaac tttataaagg atccggaacc gcaccggtct tgctagattc taatcaagag
     2161 gatgtcagaa tgccatttgc ctgagagatg caggcttcat ttttgatact tttttatttg
     2221 taacctatat agtataggat tttttttgtc attttgtttc ttctcgtacg agcttgctcc
     2281 tgatcagcct atctcgcagc tgatgaatat cttgtggtag gggtttggga aaatcattcg
     2341 agtttgatgt ttttcttggt atttcccact cctcttcaga gtacagaaga ttaagtgaga
     2401 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     2461 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     2521 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     2581 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     2641 cgtggcgctt tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     2701 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     2761 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     2821 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     2881 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
     2941 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     3001 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     3061 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     3121 catgagatca gatctgagct cgtttggccg tggccgtgct cgtcctcgtc ggccggcttg
     3181 tcgacgacgg cggtctccgt cgtcaggatc atccgggcca caagcttgct gacagaagcc
     3241 tcaagaaaaa aaaaattctt cttcgactat gctggaggca gagatgatcg agccggtagt
     3301 taactatata tagctaaatt ggttccatca cctcgagtca attcttgatt tagtatacac
     3361 ataaccaaat ttggatcaag tttgaagtaa aactttaact tcagctcctt acatttgcac
     3421 taagatctct gctactctgg tcccaagtga accacctttt ggaccctatt gaccggacct
     3481 taacttgcca aacctaaacg cttaatgcct cagacgtttt aatgcctctc aacacctcca
     3541 aggttgcttt cttgagcatg cctactaggg actttaacga actgtggggt tgcagacagt
     3601 ttcaggcgtg tcccgaccaa tatggcctac tagactctct gaaaaatcac agttttccag
     3661 tagttccgat caaattacca tcgaaatggt cccataaacg gacatttgac atccgttcct
     3721 gaattatagt cttccaccgt ggatcatggt gttccttttt ttcccaaaga atatcagcat
     3781 cccttaacta cgttaggtca gtgatgacaa tggaccaaat tgttgcaagg tttttctttt
     3841 tctttcatcg gcacatttca gcctcacatg cgactattat cgatcaatga aatccatcaa
     3901 gattgaaatc ttaaaattgc ccctttcact tgacaggatc cttttttgta gaaatgtctt
     3961 ggtgtcctcg tccaatcagg tagccatctc tgaaatatct ggctccgttg caactccgaa
     4021 cgacctgctg gcaacgtaaa attctccggg gtaaaacttt aatgtggagt aatggaacca
     4081 gaaacgtctc ttcccttctc tctccttcca ccgcccgtta ccgtccctag gaaattttac
     4141 tctgctggag agcttcttct acggccccct tgcagcaatg ctcttcccag cattacgttg
     4201 cgggtaaaac ggaggtcgtg tacccgacct agcagcccag ggatggaaaa gtcccggccg
     4261 tcgctggcaa taatagcggg cggacgcatg tcatgagatt attggaaacc accagaatcg
     4321 aatataaaag gcgaacacct ttcccaattt tggtttcccc tgacccaaag actttaaatt
     4381 taatttattt gtccctattt caatcaattg aacaactatc aaaacacact agtaaaaatg
     4441 cgcggagctc ctaagaaaaa gcgcaaagtc cggccggcat ctagacccgg ggagcgcccc
     4501 ttccagtgtc gcatttgcat gcggaacttt tcgcgccagg acaggcttga caggcatacc
     4561 cgtactcata ccggtgaaaa accgtttcag tgtcggatct gtatgcgaaa tttctcccag
     4621 aaggagcact tggcggggca tctacgtacg cacaccggcg agaagccatt ccaatgccga
     4681 atatgcatgc gcaacttcag tcgccgcgac aacctgaacc ggcacctaaa aacccacctg
     4741 aggaacatat gcggcggagg cacacctgca gctgcgtcga ctctagagga tccatctgct
     4801 ggagacatga gagctgccaa cctttggcca agcccgctca tgatcaaacg ctctaagaag
     4861 aacagcctgg ccttgtccct gacggccgac cagatggtca gtgccttgtt ggatgctgag
     4921 ccccccatac tctattccga gtatgatcct accagaccct tcagtgaagc ttcgatgatg
     4981 ggcttactga ccaacctggc agacagggag ctggttcaca tgatcaactg ggcgaagagg
     5041 gtgccaggct ttgtggattt gaccctccat gatcaggtcc accttctaga atgtgcctgg
     5101 ctagagatcc tgatgattgg tctcgtctgg cgctccatgg agcacccagt gaagctactg
     5161 tttgctccta acttgctctt ggacaggaac cagggaaaat gtgtagaggg catggtggag
     5221 atcttcgaca tgctgctggc tacatcatct cggttccgca tgatgaatct gcagggagag
     5281 gagtttgtgt gcctcaaatc tattattttg cttaattctg gagtgtacac atttctgtcc
     5341 agcaccctga agtctctgga agagaaggac catatccacc gagtcctgga caagatcaca
     5401 gacactttga tccacctgat ggccaaggca ggcctgaccc tgcagcagca gcaccagcgg
     5461 ctggcccagc tcctcctcat cctctcccac atcaggcaca tgagtaacaa aggcatggag
     5521 catctgtaca gcatgaagtg caagaacgtg gtgcccctct atgacctgct gctggagatg
     5581 ctggacgccc accgcctaca tgcgcccact agccgtggag gggcatccgt ggaggagacg
     5641 gaccaaagcc acttggccac tgcgggctct acttcatcgc ctagggccga cgcgctggac
     5701 gatttcgatc tcgacatgct gggttctgat gccctcgatg actttgacct ggatatgttg
     5761 ggaagcgacg cattggatga ctttgatctg gacatgctcg gctccgatgc tctggacgat
     5821 ttcgatctcg atatgttaat taactacccg tacgacgttc cggactacgc ttcttgaggt
     5881 accatcggta gaccggtctt gctagattct aatcaagagg atgtcagaat gccatttgcc
     5941 tgagagatgc aggcttcatt tttgatactt ttttatttgt aacctatata gtataggatt
     6001 ttttttgtca ttttgtttct tctcgtacga gcttgctcct gatcagccta tctcgcagct
     6061 gatgaatatc ttgtggtagg ggtttgggaa aatcattcga gtttgatgtt tttcttggta
     6121 tttcccactc ctcttcagag tacagaagat taagtgagag ctagcatatc atatagaagt
     6181 catcgcggca gatcaattca tcaatcggag tgaggatatt gcgagtttac caccatcaat
     6241 cggagtgagg atctccaatt ggtgacggtc cagtcatcaa tcggagtgag gattcagctg
     6301 cttctcgagg ccgcacatca atcggagtga ggatgtacag ggtgggctgt tccaccatca
     6361 atcggagtga ggatttgcgt caatggggcg gagttcatca atcggagtga ggatatcgaa
     6421 gtcatcgaga gcactcatca atcggagtga ggatatactc cacccattga cgtcacatca
     6481 atcggagtga ggattggaac cagaagggtc tcttcatcaa tcggagtgag gataccctat
     6541 gggcgcgcct aacccctact tgacagcaat atataaacag aaggaagctg ccctgtctta
     6601 aacctttttt tttatcatca ttattagctt actttcataa ttgcgactgg ttccaattga
     6661 caagcttttg attttaacga cttttaacga caacttgaga agatcaaaaa acaactaatt
     6721 attcgaaacg cgaattcatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     6781 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
     6841 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     6901 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
     6961 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     7021 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     7081 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     7141 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
     7201 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
     7261 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     7321 tgctgcccga caaccactac ctgagcaccc agagcgccct gagcaaagac cccaacgaga
     7381 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     7441 acgagctgta caaggccgcg gcggccgcca gctttctaga acaaaaactc atctcagaag
     7501 aggatctgaa tagcgccgtc gaccatcatc atcatcatca ttgaccggtc ttgctagatt
     7561 ctaatcaaga ggatgtcaga atgccatttg cctgagagat gcaggcttca tttttgatac
     7621 ttttttattt gtaacctata tagtatagga ttttttttgt cattttgttt cttctcgtac
     7681 gagcttgctc ctgatcagcc tatctcgcag ctgatgaata tcttgtggta ggggtttggg
     7741 aaaatcattc gagtttgatg tttttcttgg tatttcccac tcctcttcag agtacagaag
     7801 attaagtgag accttcgttt gtgcggatcc cccacacacc atagcttcaa aatgtttcta
     7861 ctcctttttt actcttccag attttctcgg actccgcgca tcgccgtacc acttcaaaac
     7921 acccaagcac agcatactaa attttccctc tttcttcctc tagggtgtcg ttaattaccc
     7981 gtactaaagg tttggaaaag aaaaaagaga ccgcctcgtt tctttttctt cgtcgaaaaa
     8041 ggcaataaaa atttttatca cgtttctttt tcttgaaatt ttttttttta gtttttttct
     8101 ctttcagtga cctccattga tatttaagtt aataaacggt cttcaatttc tcaagtttca
     8161 gtttcatttt tcttgttcta ttacaacttt ttttacttct tgttcattag aaagaaagca
     8221 tagcaatcta atctaagggg cggtgttgac aattaatcat cggcatagta tatcggcata
     8281 gtataatacg acaaggtgag gaactaaacc atggccaagt tgaccagtgc cgttccggtg
     8341 ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc
     8401 cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac cctgttcatc
     8461 agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc
     8521 ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca c
//
