LOCUS       sequence-189281-        4794 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GFP reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4794)
  AUTHORS   Tang W, Hu JH, Liu DR
  TITLE     Aptazyme-embedded guide RNAs enable ligand-responsive genome editing
            and transcriptional activation.
  JOURNAL   Nat Commun. 2017 Jun 28;8:15939. doi: 10.1038/ncomms15939.
  PUBMED    28656978
REFERENCE   2  (bases 1 to 4794)
  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: "2017-06-28"; volume: "8:15939. doi"; pages: " 
            10.1038/ncomms15939"
COMMENT     Sequence Label: pWT015a
FEATURES             Location/Qualifiers
     source          1..4794
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        239..257
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     239..256
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     polyA_signal    420..501
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     445..464
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1454..1477)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      1477..1486
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1500..1524)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(1521..1559)
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1682..1701)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1683..1701)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1708..1725)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1708..1724)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1717..1739)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1862..2164
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     2043..2062
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2513..3307
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2513..3307
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2567..2586)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3177..3196
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      3507..3516
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3513..3887
                     /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"
     rep_origin      4025..4613
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4514..4533
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat
      241 ttaggtgaca ctatagaata ctcaagctat gcatcaagct tggtaccgag ctcggatcca
      301 ctagtaacgg ccgccagtgt gctggaattc gccctttatt accgcctttg agtgagctga
      361 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagctcgagg
      421 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
      481 ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag
      541 ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg tatggctgat
      601 tatgatcctc tagagatatc gtcgacaagc ttatcgatgc ggccgcgcta gcacgcgtca
      661 gctgactaga ggatccccgg gtaccgagct cgaattccta gaactggcat gcatctttgt
      721 acagttcgtc cataccgtgg gtgataccag cagcggtaac gaattccagc agaaccatgt
      781 ggtcacgttt ttcgttcggg tctttagaca gaacagactg ggtagacagg tagtggttgt
      841 ccggcagcag aaccggaccg tcaccgaccg gggtgttctg ctggtagtgg tcagccagct
      901 gaacagaacc gtcttcaacg ttgtgacgga ttttgaagtt agctttgata ccgtttttct
      961 gtttgtcagc ggtgatgtaa acgttgtgag agttgaagtt gtattccagt ttgtgaccca
     1021 ggatgttacc gtcttctttg aagtcgatac ctttcagttc gatacggtta accagggtgt
     1081 caccttcgaa tttaacttca gcacgggttt cgtaggtacc gtcgtctttg aaagagatgg
     1141 tacgttcctg aacgtaacct tccggcatag cagatttgaa gaagtcgtgc tgtttcatgt
     1201 ggtccgggta acgagcgagg cactgaacac cgtaggtcag ggtggtaacc agggtcggcc
     1261 acggaaccgg cagtttaccg gtggtgcaga tgaatttcag ggtcagttta ccgttggtag
     1321 cgtcaccttc accttcacca cgaacagaga atttgtgacc gttaacgtca ccgtccagtt
     1381 caaccaggat cggaacaaca ccggtgaaca gttcttcacc tttacgggtg gaacccagtc
     1441 cgccaccgga agttttatcg tcgtcatcct tgtagtccat ggtggcgaat tctccaggcg
     1501 atctgacggt tcactaaacg agctctgctt atataggcct cccaccgtac acgcctacct
     1561 cgcccccggc aagctgcccg tgcccactat taccctgtta tccctacgtc gcaagggcga
     1621 attctgcaga tatccatcac actggcggcc gctcgagcat gcatctagag ggcccaattc
     1681 gccctatagt gagtcgtatt acaattcact ggccgtcgtt ttacaacgtc gtgactggga
     1741 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     1801 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tatacgtacg
     1861 gcagtttaag gtttacacct ataaaagaga gagccgttat cgtctgtttg tggatgtaca
     1921 gagtgatatt attgacacgc cggggcgacg gatggtgatc cccctggcca gtgcacgtct
     1981 gctgtcagat aaagtctccc gtgaacttta cccggtggtg catatcgggg atgaaagctg
     2041 gcgcatgatg accaccgata tggccagtgt gccggtctcc gttatcgggg aagaagtggc
     2101 tgatctcagc caccgcgaaa atgacatcaa aaacgccatt aacctgatgt tctggggaat
     2161 ataaatgtca ggcatgagat tatcaaaaag gatcttcacc tagatccttt tcacgtagaa
     2221 agccagtccg cagaaacggt gctgaccccg gatgaatgtc agctactggg ctatctggac
     2281 aagggaaaac gcaagcgcaa agagaaagca ggtagcttgc agtgggctta catggcgata
     2341 gctagactgg gcggttttat ggacagcaag cgaaccggaa ttgccagctg gggcgccctc
     2401 tggtaaggtt gggaagccct gcaaagtaaa ctggatggct ttcttgccgc caaggatctg
     2461 atggcgcagg ggatcaagct ctgatcaaga gacaggatga ggatcgtttc gcatgattga
     2521 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     2581 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     2641 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaagacga
     2701 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     2761 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     2821 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     2881 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     2941 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     3001 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg agcatgcccg acggcgagga
     3061 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     3121 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     3181 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     3241 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     3301 cttctgaatt attaacgctt acaatttcct gatgcggtat tttctcctta cgcatctgtg
     3361 cggtatttca caccgcatca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     3421 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     3481 tgcttcaata atagcacgtg aggagggcca ccatggccaa gttgaccagt gccgttccgg
     3541 tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg ctcgggttct
     3601 cccgggactt cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg accctgttca
     3661 tcagcgcggt ccaggaccag gtggtgccgg acaacaccct ggcctgggtg tgggtgcgcg
     3721 gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct
     3781 ccgggccggc catgaccgag atcggcgagc agccgtgggg gcgggagttc gccctgcgcg
     3841 acccggccgg caactgcgtg cacttcgtgg ccgaggagca ggactgacac gtgctaaaac
     3901 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     3961 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     4021 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     4081 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     4141 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     4201 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     4261 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     4321 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     4381 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     4441 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     4501 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     4561 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     4621 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     4681 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     4741 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaag
//
