LOCUS       sequence-147726-        7867 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  255R. Promoter: scTEF1, minimal Promoter: GAP. Figure 5. Expresses 
            GFP and RFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7867)
  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 7867)
  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: PP362
FEATURES             Location/Qualifiers
     source          1..7867
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        157..568
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        576..623
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             642..1016
                     /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      1082..1329
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1098..1116)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     promoter        1344..2282
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     terminator      3046..3292
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     rep_origin      complement(3353..3941)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3433..3452)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    4061..4110
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="attB site for the Mycopbacteriophage Bxb1 integrase 
                     (Xu et al., 2013)"
     promoter        4231..4637
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     CDS             4661..4681
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5900..6049
                     /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             6056..6082
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      6124..6370
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             join(6936..6938,6939..6941,6942..7652)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(6936..6938,6939..6941,6942..7652)
                     /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(6981..7002)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(7242..7261)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7589..7610
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             7677..7706
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             7722..7739
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      join(7763..7867,1..142)
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
ORIGIN
        1 tcttctcgta cgagcttgct cctgatcagc ctatctcgca gctgatgaat atcttgtggt
       61 aggggtttgg gaaaatcatt cgagtttgat gtttttcttg gtatttccca ctcctcttca
      121 gagtacagaa gattaagtga gaccttcgtt tgtgcggatc ccccacacac catagcttca
      181 aaatgtttct actccttttt tactcttcca gattttctcg gactccgcgc atcgccgtac
      241 cacttcaaaa cacccaagca cagcatacta aattttccct ctttcttcct ctagggtgtc
      301 gttaattacc cgtactaaag gtttggaaaa gaaaaaagag accgcctcgt ttctttttct
      361 tcgtcgaaaa aggcaataaa aatttttatc acgtttcttt ttcttgaaat tttttttttt
      421 agtttttttc tctttcagtg acctccattg atatttaagt taataaacgg tcttcaattt
      481 ctcaagtttc agtttcattt ttcttgttct attacaactt tttttacttc ttgttcatta
      541 gaaagaaagc atagcaatct aatctaaggg gcggtgttga caattaatca tcggcatagt
      601 atatcggcat agtataatac gacaaggtga ggaactaaac catggccaag ttgaccagtg
      661 ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc
      721 tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga
      781 ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt
      841 gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc
      901 gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg
      961 ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgacacg
     1021 tccgacggcg gcccacgggt cccaggcctc ggagatccgt cccccttttc ctttgtcgat
     1081 atcatgtaat tagttatgtc acgcttacat tcacgccctc cccccacatc cgctctaacc
     1141 gaaaaggaag gagttagaca acctgaagtc taggtcccta tttatttttt tatagttatg
     1201 ttagtattaa gaacgttatt tatatttcaa atttttcttt tttttctgta cagacgcgtg
     1261 tacgcatgta acattatact gaaaaccttg cttgagaagg ttttgggacg ctcgaaggct
     1321 ttaatttgca agctggagac caagatctaa catccaaaga cgaaaggttg aatgaaacct
     1381 ttttgccatc cgacatccac aggtccattc tcacacataa gtgccaaacg caacaggagg
     1441 ggatacacta gcagcagacc gttgcaaacg caggacctcc actcctcttc tcctcaacac
     1501 ccacttttgc catcgaaaaa ccagcccagt tattgggctt gattggagct cgctcattcc
     1561 aattccttct attaggctac taacaccatg actttattag cctgtctatc ctggcccccc
     1621 tggcgaggtt catgtttgtt tatttccgaa tgcaacaagc tccgcattac acccgaacat
     1681 cactccagat gagggctttc tgagtgtggg gtcaaatagt ttcatgttcc ccaaatggcc
     1741 caaaactgac agtttaaacg ctgtcttgga acctaatatg acaaaagcgt gatctcatcc
     1801 aagatgaact aagtttggtt cgttgaaatg ctaacggcca gttggtcaaa aagaaacttc
     1861 caaaagtcgg cataccgttt gtcttgtttg gtattgattg acgaatgctc aaaaataatc
     1921 tcattaatgc ttagcgcagt ctctctatcg cttctgaacc ccggtgcacc tgtgccgaaa
     1981 cgcaaatggg gaaacacccg ctttttggat gattatgcat tgtctccaca ttgtatgctt
     2041 ccaagattct ggtgggaata ctgctgatag cctaacgttc atgatcaaaa tttaactgtt
     2101 ctaaccccta cttgacagca atatataaac agaaggaagc tgccctgtct taaacctttt
     2161 tttttatcat cattattagc ttactttcat aattgcgact ggttccaatt gacaagcttt
     2221 tgattttaac gacttttaac gacaacttga gaagatcaaa aaacaactaa ttattcgaaa
     2281 cgaattcatg gcaacttccg gtatggtctc aaagggagag gaaaataata tggctattat
     2341 taaggagttt atgcgtttta aggtacatat ggaaggttct gtcaacggtc acgaattcga
     2401 aattgaaggt gagggggagg ggagaccata cgagggaact cagactgcta agttaaaggt
     2461 cactaaaggt ggtcctttac ctttcgcctg ggatatcctg tctccacagt ttatgtacgg
     2521 ttcaaaggct tatgtgaaac atcctgccga tatcccagat tatcttaaac tttctttccc
     2581 tgagggtttt aagtgggaga gggtaatgaa ctttgaagac ggtggtgtgg tcactgttac
     2641 tcaggactca agtctgcagg acggtgagtt catctacaag gtgaagctga gaggtaccaa
     2701 ttttccatca gatggtcccg tgatgcaaaa aaagacaatg ggttgggaag cttctagtga
     2761 acgtatgtat cccgaagatg gagctttgaa aggtgaaatt aagcaaagac taaaacttaa
     2821 ggatggtgga cattacgatg ctgaagttaa gacgacctac aaggccaaaa agccagtcca
     2881 gttgcctgga gcatacaatg ttaacatcaa attggatata acttcccata atgaagacta
     2941 taccatcgtc gagcaatacg aacgagccga agggagacac agtactggtg gtatggatga
     3001 actttataaa ggatccggaa ccgcaccggt cttgctagat tctaatcaag aggatgtcag
     3061 aatgccattt gcctgagaga tgcaggcttc atttttgata cttttttatt tgtaacctat
     3121 atagtatagg attttttttg tcattttgtt tcttctcgta cgagcttgct cctgatcagc
     3181 ctatctcgca gctgatgaat atcttgtggt aggggtttgg gaaaatcatt cgagtttgat
     3241 gtttttcttg gtatttccca ctcctcttca gagtacagaa gattaagtga gacatgtgag
     3301 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     3361 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     3421 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     3481 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     3541 tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     3601 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     3661 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     3721 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     3781 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     3841 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     3901 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     3961 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     4021 cagatctgag ctcgtttggc cgtggccgtg ctcgtcctcg tcggccggct tgtcgacgac
     4081 ggcggtctcc gtcgtcagga tcatccgggc cacaagcttg ctgacagaag cctcaagaaa
     4141 aaaaaaattc ttcttcgact atgctggagg cagagatgat cgagccggta gttaactata
     4201 tatagctaaa ttggttccat cacctcgagt ccacacacca tagcttcaaa atgtttctac
     4261 tcctttttta ctcttccaga ttttctcgga ctccgcgcat cgccgtacca cttcaaaaca
     4321 cccaagcaca gcatactaaa tttcccctct ttcttcctct agggtgtcgt taattacccg
     4381 tactaaaggt ttggaaaaga aaaaagagac cgcctcgttt ctttttcttc gtcgaaaaag
     4441 gcaataaaaa tttttatcac gtttcttttt cttgaaaatt ttttttttga tttttttctc
     4501 tttcgatgac ctcccattga tatttaagtt aataaacggt cttcaatttc tcaagtttca
     4561 gtttcatttt tcttgttcta ttacaacttt ttttacttct tgctcattag aaagaaagca
     4621 tagcaatcta atctaaggac tagtaaaaat gcgcggagct cctaagaaaa agcgcaaagt
     4681 ccggccggca tctagacccg gggagcgccc cttccagtgt cgcatttgca tgcggaactt
     4741 ttcgcgccag gacaggcttg acaggcatac ccgtactcat accggtgaaa aaccgtttca
     4801 gtgtcggatc tgtatgcgaa atttctccca gaaggagcac ttggcggggc atctacgtac
     4861 gcacaccggc gagaagccat tccaatgccg aatatgcatg cgcaacttca gtcgccgcga
     4921 caacctgaac cggcacctaa aaacccacct gaggaacata tgcggcggag gcacacctgc
     4981 agctgcgtcg actctagagg atccatctgc tggagacatg agagctgcca acctttggcc
     5041 aagcccgctc atgatcaaac gctctaagaa gaacagcctg gccttgtccc tgacggccga
     5101 ccagatggtc agtgccttgt tggatgctga gccccccata ctctattccg agtatgatcc
     5161 taccagaccc ttcagtgaag cttcgatgat gggcttactg accaacctgg cagacaggga
     5221 gctggttcac atgatcaact gggcgaagag ggtgccaggc tttgtggatt tgaccctcca
     5281 tgatcaggtc caccttctag aatgtgcctg gctagagatc ctgatgattg gtctcgtctg
     5341 gcgctccatg gagcacccag tgaagctact gtttgctcct aacttgctct tggacaggaa
     5401 ccagggaaaa tgtgtagagg gcatggtgga gatcttcgac atgctgctgg ctacatcatc
     5461 tcggttccgc atgatgaatc tgcagggaga ggagtttgtg tgcctcaaat ctattatttt
     5521 gcttaattct ggagtgtaca catttctgtc cagcaccctg aagtctctgg aagagaagga
     5581 ccatatccac cgagtcctgg acaagatcac agacactttg atccacctga tggccaaggc
     5641 aggcctgacc ctgcagcagc agcaccagcg gctggcccag ctcctcctca tcctctccca
     5701 catcaggcac atgagtaaca aaggcatgga gcatctgtac agcatgaagt gcaagaacgt
     5761 ggtgcccctc tatgacctgc tgctggagat gctggacgcc caccgcctac atgcgcccac
     5821 tagccgtgga ggggcatccg tggaggagac ggaccaaagc cacttggcca ctgcgggctc
     5881 tacttcatcg cctagggccg acgcgctgga cgatttcgat ctcgacatgc tgggttctga
     5941 tgccctcgat gactttgacc tggatatgtt gggaagcgac gcattggatg actttgatct
     6001 ggacatgctc ggctccgatg ctctggacga tttcgatctc gatatgttaa ttaactaccc
     6061 gtacgacgtt ccggactacg cttcttgagg taccatcggt agaccggtct tgctagattc
     6121 taatcaagag gatgtcagaa tgccatttgc ctgagagatg caggcttcat ttttgatact
     6181 tttttatttg taacctatat agtataggat tttttttgtc attttgtttc ttctcgtacg
     6241 agcttgctcc tgatcagcct atctcgcagc tgatgaatat cttgtggtag gggtttggga
     6301 aaatcattcg agtttgatgt ttttcttggt atttcccact cctcttcaga gtacagaaga
     6361 ttaagtgaga gctagcatat catatagaag tcatcgcggc agatcaattc atcaatcgga
     6421 gtgaggatat tgcgagttta ccaccatcaa tcggagtgag gatctccaat tggtgacggt
     6481 ccagtcatca atcggagtga ggattcagct gcttctcgag gccgcacatc aatcggagtg
     6541 aggatgtaca gggtgggctg ttccaccatc aatcggagtg aggatttgcg tcaatggggc
     6601 ggagttcatc aatcggagtg aggatatcga agtcatcgag agcactcatc aatcggagtg
     6661 aggatatact ccacccattg acgtcacatc aatcggagtg aggattggaa ccagaagggt
     6721 ctcttcatca atcggagtga ggatacccta tgggcgcgcc gtcgctggca ataatagcgg
     6781 gcggacgcat gtcatgagat tattggaaac caccagaatc gaatataaaa ggcgaacacc
     6841 tttcccaatt ttggtttccc ctgacccaaa gactttaaat ttaatttatt tgtccctatt
     6901 tcaatcaatt gaacaactat caaaacacag aattcatggt gagcaagggc gaggagctgt
     6961 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
     7021 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct
     7081 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg
     7141 tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
     7201 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
     7261 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     7321 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc
     7381 acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc
     7441 gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca
     7501 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag agcgccctga
     7561 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
     7621 ggatcactct cggcatggac gagctgtaca aggccgcggc ggccgccagc tttctagaac
     7681 aaaaactcat ctcagaagag gatctgaata gcgccgtcga ccatcatcat catcatcatt
     7741 gaccggtctt gctagattct aatcaagagg atgtcagaat gccatttgcc tgagagatgc
     7801 aggcttcatt tttgatactt ttttatttgt aacctatata gtataggatt ttttttgtca
     7861 ttttgtt
//
