LOCUS       sequence-222989-        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"
     primer_bind     368..387
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(670..1258)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(750..769)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    1374..1657
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
     terminator      complement(1686..1732)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(join(1906..2619,2620..2622,2623..2625))
                     /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(1909..2619,2620..2622,2623..2625))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1951..1972)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2300..2319
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2559..2580
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     misc_feature    join(2626..2634,2635..2637,2638..3212)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2799..2818)
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     3028..3045
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     complement(3259..3278)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3260..3278)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        3304..3403
                     /label=CMVd1 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields slightly reduced expression 
                     relative to the full-length promoter"
     CDS             join(3447..3449,3450..3770)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     promoter        5967..6066
                     /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(6986..7004)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    7041..7162
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7052..7071)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     7106..7125
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             join(7561..7857,1..498)
                     /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(7615..7634)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
       61 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
      121 cgcatcgagc gagcacgcac tcggatggaa gccggtcttg tcgatcagga tgatctggac
      181 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgtatgccg
      241 gatggtgagg atctcgtcgt gactcatggc gatgcctgct tgccgaatat catggtggaa
      301 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
      361 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
      421 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
      481 cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca
      541 accggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
      601 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
      661 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
      721 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
      781 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
      841 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
      901 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
      961 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     1021 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     1081 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
     1141 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     1201 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatttcaaga
     1261 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     1321 gattttggtc atgagattat caaaaaggat cttcacctag atccttttat agtccggaaa
     1381 tacaggaacg cacgctggat ggcccttcgc tgggatggtg aaaccatgaa aaatggcagc
     1441 ttcagtggat taagtggggg taatgtggcc tgtaccctct ggttgcatag gtattcatac
     1501 ggttaaaatt tatcaggcgc gattgcggca gtttttcggg tggtttgttg ccatttttac
     1561 ctgtctgctg ccgtgatcgc gctgaacgcg ttttagcggt gcgtacaatt aagggattat
     1621 ggtaaatcca cttactgtct gccctcgtag ccatcgagat aaaccgcagt actccggcca
     1681 cgatgcaaaa aacccctcaa gacccgttta gaggccccaa ggggttatgc tatcagaagc
     1741 tttcatttca gcttcagttt tcagatgttt gaaacgatcg aatttttcca gagtttccgg
     1801 atgagatttg aacagtcgaa tcaagatgtc ctgaccatga ccagcgacgt cagcttcaac
     1861 tttagcccaa acatgcagaa ccagctgcca ttcaccttca gcgctttact tgtacagctc
     1921 gtccatgccg agagtgatcc cggcggcggt cacgaactcc agcaggacca tgtgatcgcg
     1981 cttctcgttg gggtctttgc tcagggcgga ctgggtgctc aggtagtggt tgtcgggcag
     2041 cagcacgggg ccgtcgccga tgggggtgtt ctgctggtag tggtcggcga gctgcacgct
     2101 gccgtcctcg atgttgtggc ggatcttgaa gttcaccttg atgccgttct tctgcttgtc
     2161 ggccatgata tagacgttgt ggctgttgta gttgtactcc agcttgtgcc ccaggatgtt
     2221 gccgtcctcc ttgaagtcga tgcccttcag ctcgatgcgg ttcaccaggg tgtcgccctc
     2281 gaacttcacc tcggcgcggg tcttgtagtt gccgtcgtcc ttgaagaaga tggtgcgctc
     2341 ctggacgtag ccttcgggca tggcggactt gaagaagtcg tgctgcttca tgtggtcggg
     2401 gtagcggctg aagcactgca cgccgtaggt cagggtggtc acgagggtgg gccagggcac
     2461 gggcagcttg ccggtggtgc agatgaactt cagggtcagc ttgccgtagg tggcatcgcc
     2521 ctcgccctcg ccggacacgc tgaacttgtg gccgtttacg tcgccgtcca gctcgaccag
     2581 gatgggcacc accccggtga acagctcctc gcccttgctc accatggttg tggccatatt
     2641 atcatcgtgt ttttcaaagg aaaaccacgt ccccgtggtt cggggggcct agacgttttt
     2701 ttaacctcga ctaaacacat gtaaagcatg tgtaccgagg ccccagatca gatcccatac
     2761 aatggggtac cttctgggca tccttcagcc ccttgttgaa tacgcttgag gagagccatt
     2821 tgactctttc cacaactatc caactcacaa cgtggcactg gggttgtgcc gcctttgcag
     2881 gtgtatctta tacacgtggc ttttggccgc agaggcacct gtcgccaggt ggggggttcc
     2941 gctgcctgca aagggtcgct acagacgttg tttgtcttca agaagcttcc agaggaactg
     3001 cttccttcac gacattcaac agaccttgca ttcctttggc gagaggggaa agacccctag
     3061 gaatgctcgt caagaagaca gggccaggtt tccgggccct cacattgcca aaagacggca
     3121 atatggtgga aaataacata tagacaaacg cacaccggcc ttattccaag cggcttcggc
     3181 cagtaacgtt aggggggggg gagggagagg ggcggatccg ggccctctag atgcggccgc
     3241 atgcataagc ttgggtctcc ctatagtgag tcgtattacg tccggcgtag aggatcgaga
     3301 tctccaaaat caacgggact ttccaaaatg tcgtaataac cccgccccgt tgacgcaaat
     3361 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag tgaaccgtca
     3421 gatcactaga agcttacgct accaccatgg gagtgcaggt ggaaaccatc tccccaggag
     3481 acgggcgcac cttccccaag cgcggccaga cctgcgtggt gcactacacc gggatgcttg
     3541 aagatggaaa gaaatttgat tcctcccggg acagaaacaa gccctttaag tttatgctag
     3601 gcaagcagga ggtgatccga ggctgggaag aaggggttgc ccagatgagt gtgggtcaga
     3661 gagccaaact gactatatct ccagattatg cctatggtgc cactgggcac ccaggcatca
     3721 tcccaccaca tgccactctc gtcttcgatg tggagcttct aaaactggaa acgtcgggtg
     3781 gaagcggtaa agcatttatg caagttgtcg aggctgacat gctctctaag ggtctactcg
     3841 gtggcgaggc gtggtcttcg tggcataagg aagactctat tcatgtagga gtacgctgca
     3901 tcgagatgct cattgagtca accggaatgg ttagcttaca ccgccaaaat gctggcgtag
     3961 taggtcaaga ctctgagact atcgaactcg cacctgaata cgctgaggct atcgcaaccc
     4021 gtgcaggtgc gctggctggc atctctccga tgttccaacc ttgcgtagtt cctcctaagc
     4081 cgtggactgg cattactggt ggtggctatt gggctaacgg tcgtcgtcct ctggcgctgg
     4141 tgcgtactca cagtaagaaa gcactgatgc gctacgaaga cgtttacatg cctgaggtgt
     4201 acaaagcgat taacattgcg caaaacaccg catggaaaat caacaagaaa gtcctagcgg
     4261 tcgccaacgt aatcaccaag tggaagcatt gtccggtcga ggacatccct gcgattgagc
     4321 gtgaagaact cccgatgaaa ccggaagaca tcgacatgaa tcctgaggct ctcaccgcgt
     4381 ggaaacgtgc tgccgctgct gtgtaccgca aggacaaggc tcgcaagtct cgccgtatca
     4441 gccttgagtt catgcttgag caagccaata agtttgctaa ccataaggcc atctggttcc
     4501 cttacaacat ggactggcgc ggtcgtgttt acgctgtgtc aatgttcaac ccgcaaggta
     4561 acgatatgac caaaggactg cttacgctgg cgaaaggtaa accaatcggt aaggaaggtt
     4621 actactggct gaaaatccac ggtgcaaact gtgcgggtgt cgataaggtt ccgttccctg
     4681 agcgcatcaa gttcattgag gaaaaccacg agaacatcat ggcttgcgct aagtctccac
     4741 tggagaacac ttggtgggct gagcaagatt ctccgttctg cttccttgcg ttctgctttg
     4801 agtacgctgg ggtacagcac cacggcctga gctataactg ctcccttccg ctggcgtttg
     4861 acgggtcttg ctctggcatc cagcacttct ccgcgatgct ccgagatgag gtaggtggtc
     4921 gcgcggttaa cttgcttcct agtgaaaccg ttcaggacat ctacgggatt gttgctaaga
     4981 aagtcaacga gattctacaa gcagacgcaa tcaatgggac cgataacgaa gtagttaccg
     5041 tgaccgatga gaacactggt gaaatctctg agaaagtcaa gctgggcact aaggcactgg
     5101 ctggtcaatg gctggcttac ggtgttactc gcagtgtgac taagcgttca gtcatgacgc
     5161 tggcttacgg gtccaaagag ttcggcttcc gtcaacaagt gctggaagat accattcagc
     5221 cagctattga ttccggcaag ggtctgatgt tcactcagcc gaatcaggct gctggataca
     5281 tggctaagct gatttgggaa tctgtgagcg tgacggtggt agctgcggtt gaagcaatga
     5341 actggcttaa gtctgctgct aagctgctgg ctgctgaggt caaagataag aagactggag
     5401 agattcttcg caagcgttgc gctgtgcatt gggtaactcc tgatggtttc cctgtgtggc
     5461 aggaatacaa gaagcctatt cagacgcgct tgaacctgat gttcctcggt cagttccgct
     5521 tacagcctac cattaacacc aacaaagata gcgagattga tgcacacaaa caggagtctg
     5581 gtatcgctcc taactttgta cacagccaag acggtagcca ccttcgtaag actgtagtgt
     5641 gggcacacga gaagtacgga atcgaatctt ttgcactgat tcacgactcc ttcggtacca
     5701 ttccggctga cgctgcgaac ctgttcaaag cagtgcgcga aactatggtt gacacatatg
     5761 agtcttgtga tgtactggct gatttctacg accagttcgc tgaccagttg cacgagtctc
     5821 aattggacaa aatgccagca cttccggcta aaggtaactt gaacctccgt gacatcttag
     5881 agtcggactt cgcgttcgcg taatgtttaa acgaattcgg gctcggtacc cggggatcct
     5941 ctagagtcga cctgcaggca tgcaagccaa aatcaacggg actttccaaa atgtcgtaat
     6001 aaccccgccc cgttgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
     6061 agagctggtt tagtgaaccg tcagatcact agaagcttac gctaccacca tgaacacgat
     6121 taacatcgct aagaacgact tctctgacat cgaactggct gctatcccgt tcaacactct
     6181 ggctgaccat tacggtgagc gttcagctcg cggacagttg gcccttgagc atgagtctta
     6241 cgagatgggt gaagcacgct tccgcaagat gtttgagcgt caacttaaag ctggtgaggt
     6301 tgcggataac gctgccgcca agcctctcat cactacccta ctccctaaga tgattgcccg
     6361 catcaacgac tggtttgagg aagtgaaagc taagcgcggc aggcgcccga cagccttcca
     6421 gttcctaaaa gaaatcaagc cggaagccgt agcgtacatc accattaaga cctctctggc
     6481 ttgcctaacc agtgctgaca atacaaccgt tcaggctgta gcaagcgcaa tcggtcggac
     6541 cattgaggac gaggctcgct tcggtcgtat ccgtgacctt gaagctaagc acttcaagaa
     6601 aaacgttgag gaacaactca acaagcgcgt agggcacgtc tacaagggtg gctctggctc
     6661 tggctcgagc gcggtgcttt ggcatgagca atggtatgag ggtctggatg acgccagtag
     6721 gcagtttttt ggagaacata ataccgaaaa aatgtttgct gctttagagc ctctgtacga
     6781 aatgctgaag agaggaccgg aaactttgag ggaaatatcg ttccaaaatt cttttggtag
     6841 ggacttgaat gacgcttacg aatggctgat gaattacaaa aaatctaaag atgttagtaa
     6901 tttaaaccaa gcgtgggaca tttactataa tgttttcagg aaaattggtt aaaagcccga
     6961 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcg
     7021 gccgcttcga gcagacatga taagatacat tgatgagttt ggacaaacca caactagaat
     7081 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     7141 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca
     7201 gggggagatg tgggaggttt ttttaagcaa gtaaaacctc tacaaatgtg gtaaaatcga
     7261 attctaatgg atcctctttg cgcttgcgtt ttcccttgtc cagatagccc agtagctgac
     7321 attcatccgg ggtcagcacc gtttctgcgg actggctttc tacgtgttcc gcttccttta
     7381 gcagcccttg cgccctgagt gcttgcggca gcgtgagctt caaaagaatt gccagctggg
     7441 gcgccctctg gtaaggttgg gaagccctgc aaagtaaact ggatggcttt cttgccgcca
     7501 aggatctgat ggcgcagggg atcaagatct gatcaagaga caggatgacg gtcgtttcgc
     7561 atgcttgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     7621 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     7681 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     7741 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     7801 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccgggg
//
