LOCUS       sequence-227368-        7857 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  rapa-T7- mRNA(GFP).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7857)
  AUTHORS   Pu J, Zinkus-Boltz J, Dickinson BC
  TITLE     Evolution of a split RNA polymerase as a versatile biosensor 
            platform.
  JOURNAL   Nat Chem Biol. 2017 Apr;13(4):432-438. doi: 10.1038/nchembio.2299. 
            Epub 2017 Feb 13.
  PUBMED    28192413
REFERENCE   2  (bases 1 to 7857)
  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"; doi: 
            "10.1038/nchembio.2299"; journalName: "Nat Chem Biol"; date: 
            "2017-04"; volume: "13"; issue: "4"; pages: "432-438"
COMMENT     Sequence Label: p6-8
FEATURES             Location/Qualifiers
     source          1..7857
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..100
                     /label=CMVd1 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields slightly reduced expression 
                     relative to the full-length promoter"
     CDS             join(144..146,147..467)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     promoter        2664..2763
                     /label=CMVd1 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields slightly reduced expression 
                     relative to the full-length promoter"
     primer_bind     complement(3683..3701)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    3738..3859
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3749..3768)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3803..3822
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             4258..5052
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MLEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4312..4331)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4922..4941
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(5224..5812)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5304..5323)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    5928..6211
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
     terminator      complement(6240..6286)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(join(6460..7173,7174..7176,7177..7179))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(6463..7173,7174..7176,7177..7179))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6505..6526)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     6854..6873
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7113..7134
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     misc_feature    join(7180..7188,7189..7191,7192..7766)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(7353..7372)
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     7582..7599
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     complement(7813..7832)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7814..7832)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ccaaaatcaa cgggactttc caaaatgtcg taataacccc gccccgttga cgcaaatggg
       61 cggtaggcgt gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat
      121 cactagaagc ttacgctacc accatgggag tgcaggtgga aaccatctcc ccaggagacg
      181 ggcgcacctt ccccaagcgc ggccagacct gcgtggtgca ctacaccggg atgcttgaag
      241 atggaaagaa atttgattcc tcccgggaca gaaacaagcc ctttaagttt atgctaggca
      301 agcaggaggt gatccgaggc tgggaagaag gggttgccca gatgagtgtg ggtcagagag
      361 ccaaactgac tatatctcca gattatgcct atggtgccac tgggcaccca ggcatcatcc
      421 caccacatgc cactctcgtc ttcgatgtgg agcttctaaa actggaaacg tcgggtggaa
      481 gcggtaaagc atttatgcaa gttgtcgagg ctgacatgct ctctaagggt ctactcggtg
      541 gcgaggcgtg gtcttcgtgg cataaggaag actctattca tgtaggagta cgctgcatcg
      601 agatgctcat tgagtcaacc ggaatggtta gcttacaccg ccaaaatgct ggcgtagtag
      661 gtcaagactc tgagactatc gaactcgcac ctgaatacgc tgaggctatc gcaacccgtg
      721 caggtgcgct ggctggcatc tctccgatgt tccaaccttg cgtagttcct cctaagccgt
      781 ggactggcat tactggtggt ggctattggg ctaacggtcg tcgtcctctg gcgctggtgc
      841 gtactcacag taagaaagca ctgatgcgct acgaagacgt ttacatgcct gaggtgtaca
      901 aagcgattaa cattgcgcaa aacaccgcat ggaaaatcaa caagaaagtc ctagcggtcg
      961 ccaacgtaat caccaagtgg aagcattgtc cggtcgagga catccctgcg attgagcgtg
     1021 aagaactccc gatgaaaccg gaagacatcg acatgaatcc tgaggctctc accgcgtgga
     1081 aacgtgctgc cgctgctgtg taccgcaagg acaaggctcg caagtctcgc cgtatcagcc
     1141 ttgagttcat gcttgagcaa gccaataagt ttgctaacca taaggccatc tggttccctt
     1201 acaacatgga ctggcgcggt cgtgtttacg ctgtgtcaat gttcaacccg caaggtaacg
     1261 atatgaccaa aggactgctt acgctggcga aaggtaaacc aatcggtaag gaaggttact
     1321 actggctgaa aatccacggt gcaaactgtg cgggtgtcga taaggttccg ttccctgagc
     1381 gcatcaagtt cattgaggaa aaccacgaga acatcatggc ttgcgctaag tctccactgg
     1441 agaacacttg gtgggctgag caagattctc cgttctgctt ccttgcgttc tgctttgagt
     1501 acgctggggt acagcaccac ggcctgagct ataactgctc ccttccgctg gcgtttgacg
     1561 ggtcttgctc tggcatccag cacttctccg cgatgctccg agatgaggta ggtggtcgcg
     1621 cggttaactt gcttcctagt gaaaccgttc aggacatcta cgggattgtt gctaagaaag
     1681 tcaacgagat tctacaagca gacgcaatca atgggaccga taacgaagta gttaccgtga
     1741 ccgatgagaa cactggtgaa atctctgaga aagtcaagct gggcactaag gcactggctg
     1801 gtcaatggct ggcttacggt gttactcgca gtgtgactaa gcgttcagtc atgacgctgg
     1861 cttacgggtc caaagagttc ggcttccgtc aacaagtgct ggaagatacc attcagccag
     1921 ctattgattc cggcaagggt ctgatgttca ctcagccgaa tcaggctgct ggatacatgg
     1981 ctaagctgat ttgggaatct gtgagcgtga cggtggtagc tgcggttgaa gcaatgaact
     2041 ggcttaagtc tgctgctaag ctgctggctg ctgaggtcaa agataagaag actggagaga
     2101 ttcttcgcaa gcgttgcgct gtgcattggg taactcctga tggtttccct gtgtggcagg
     2161 aatacaagaa gcctattcag acgcgcttga acctgatgtt cctcggtcag ttccgcttac
     2221 agcctaccat taacaccaac aaagatagcg agattgatgc acacaaacag gagtctggta
     2281 tcgctcctaa ctttgtacac agccaagacg gtagccacct tcgtaagact gtagtgtggg
     2341 cacacgagaa gtacggaatc gaatcttttg cactgattca cgactccttc ggtaccattc
     2401 cggctgacgc tgcgaacctg ttcaaagcag tgcgcgaaac tatggttgac acatatgagt
     2461 cttgtgatgt actggctgat ttctacgacc agttcgctga ccagttgcac gagtctcaat
     2521 tggacaaaat gccagcactt ccggctaaag gtaacttgaa cctccgtgac atcttagagt
     2581 cggacttcgc gttcgcgtaa tgtttaaacg aattcgggct cggtacccgg ggatcctcta
     2641 gagtcgacct gcaggcatgc aagccaaaat caacgggact ttccaaaatg tcgtaataac
     2701 cccgccccgt tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga
     2761 gctggtttag tgaaccgtca gatcactaga agcttacgct accaccatga acacgattaa
     2821 catcgctaag aacgacttct ctgacatcga actggctgct atcccgttca acactctggc
     2881 tgaccattac ggtgagcgtt cagctcgcgg acagttggcc cttgagcatg agtcttacga
     2941 gatgggtgaa gcacgcttcc gcaagatgtt tgagcgtcaa cttaaagctg gtgaggttgc
     3001 ggataacgct gccgccaagc ctctcatcac taccctactc cctaagatga ttgcccgcat
     3061 caacgactgg tttgaggaag tgaaagctaa gcgcggcagg cgcccgacag ccttccagtt
     3121 cctaaaagaa atcaagccgg aagccgtagc gtacatcacc attaagacct ctctggcttg
     3181 cctaaccagt gctgacaata caaccgttca ggctgtagca agcgcaatcg gtcggaccat
     3241 tgaggacgag gctcgcttcg gtcgtatccg tgaccttgaa gctaagcact tcaagaaaaa
     3301 cgttgaggaa caactcaaca agcgcgtagg gcacgtctac aagggtggct ctggctctgg
     3361 ctcgagcgcg gtgctttggc atgagcaatg gtatgagggt ctggatgacg ccagtaggca
     3421 gttttttgga gaacataata ccgaaaaaat gtttgctgct ttagagcctc tgtacgaaat
     3481 gctgaagaga ggaccggaaa ctttgaggga aatatcgttc caaaattctt ttggtaggga
     3541 cttgaatgac gcttacgaat ggctgatgaa ttacaaaaaa tctaaagatg ttagtaattt
     3601 aaaccaagcg tgggacattt actataatgt tttcaggaaa attggttaaa agcccgaaag
     3661 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcggcc
     3721 gcttcgagca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca
     3781 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat
     3841 aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg
     3901 ggagatgtgg gaggtttttt taagcaagta aaacctctac aaatgtggta aaatcgaatt
     3961 ctaatggatc ctctttgcgc ttgcgttttc ccttgtccag atagcccagt agctgacatt
     4021 catccggggt cagcaccgtt tctgcggact ggctttctac gtgttccgct tcctttagca
     4081 gcccttgcgc cctgagtgct tgcggcagcg tgagcttcaa aagaattgcc agctggggcg
     4141 ccctctggta aggttgggaa gccctgcaaa gtaaactgga tggctttctt gccgccaagg
     4201 atctgatggc gcaggggatc aagatctgat caagagacag gatgacggtc gtttcgcatg
     4261 cttgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     4321 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     4381 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag
     4441 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     4501 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     4561 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     4621 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     4681 gagcgagcac gcactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     4741 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgtat gccggatggt
     4801 gaggatctcg tcgtgactca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     4861 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     4921 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     4981 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     5041 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaaccggt
     5101 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     5161 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     5221 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     5281 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     5341 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     5401 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     5461 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     5521 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     5581 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     5641 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
     5701 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     5761 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatttc aagaagatcc
     5821 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     5881 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttatagtccg gaaatacagg
     5941 aacgcacgct ggatggccct tcgctgggat ggtgaaacca tgaaaaatgg cagcttcagt
     6001 ggattaagtg ggggtaatgt ggcctgtacc ctctggttgc ataggtattc atacggttaa
     6061 aatttatcag gcgcgattgc ggcagttttt cgggtggttt gttgccattt ttacctgtct
     6121 gctgccgtga tcgcgctgaa cgcgttttag cggtgcgtac aattaaggga ttatggtaaa
     6181 tccacttact gtctgccctc gtagccatcg agataaaccg cagtactccg gccacgatgc
     6241 aaaaaacccc tcaagacccg tttagaggcc ccaaggggtt atgctatcag aagctttcat
     6301 ttcagcttca gttttcagat gtttgaaacg atcgaatttt tccagagttt ccggatgaga
     6361 tttgaacagt cgaatcaaga tgtcctgacc atgaccagcg acgtcagctt caactttagc
     6421 ccaaacatgc agaaccagct gccattcacc ttcagcgctt tacttgtaca gctcgtccat
     6481 gccgagagtg atcccggcgg cggtcacgaa ctccagcagg accatgtgat cgcgcttctc
     6541 gttggggtct ttgctcaggg cggactgggt gctcaggtag tggttgtcgg gcagcagcac
     6601 ggggccgtcg ccgatggggg tgttctgctg gtagtggtcg gcgagctgca cgctgccgtc
     6661 ctcgatgttg tggcggatct tgaagttcac cttgatgccg ttcttctgct tgtcggccat
     6721 gatatagacg ttgtggctgt tgtagttgta ctccagcttg tgccccagga tgttgccgtc
     6781 ctccttgaag tcgatgccct tcagctcgat gcggttcacc agggtgtcgc cctcgaactt
     6841 cacctcggcg cgggtcttgt agttgccgtc gtccttgaag aagatggtgc gctcctggac
     6901 gtagccttcg ggcatggcgg acttgaagaa gtcgtgctgc ttcatgtggt cggggtagcg
     6961 gctgaagcac tgcacgccgt aggtcagggt ggtcacgagg gtgggccagg gcacgggcag
     7021 cttgccggtg gtgcagatga acttcagggt cagcttgccg taggtggcat cgccctcgcc
     7081 ctcgccggac acgctgaact tgtggccgtt tacgtcgccg tccagctcga ccaggatggg
     7141 caccaccccg gtgaacagct cctcgccctt gctcaccatg gttgtggcca tattatcatc
     7201 gtgtttttca aaggaaaacc acgtccccgt ggttcggggg gcctagacgt ttttttaacc
     7261 tcgactaaac acatgtaaag catgtgtacc gaggccccag atcagatccc atacaatggg
     7321 gtaccttctg ggcatccttc agccccttgt tgaatacgct tgaggagagc catttgactc
     7381 tttccacaac tatccaactc acaacgtggc actggggttg tgccgccttt gcaggtgtat
     7441 cttatacacg tggcttttgg ccgcagaggc acctgtcgcc aggtgggggg ttccgctgcc
     7501 tgcaaagggt cgctacagac gttgtttgtc ttcaagaagc ttccagagga actgcttcct
     7561 tcacgacatt caacagacct tgcattcctt tggcgagagg ggaaagaccc ctaggaatgc
     7621 tcgtcaagaa gacagggcca ggtttccggg ccctcacatt gccaaaagac ggcaatatgg
     7681 tggaaaataa catatagaca aacgcacacc ggccttattc caagcggctt cggccagtaa
     7741 cgttaggggg gggggaggga gaggggcgga tccgggccct ctagatgcgg ccgcatgcat
     7801 aagcttgggt ctccctatag tgagtcgtat tacgtccggc gtagaggatc gagatct
//
