LOCUS       sequence-189280-        4800 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  RFP reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4800)
  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 4800)
  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: pWT032a
FEATURES             Location/Qualifiers
     source          1..4800
                     /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(728..1429)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="SKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQ
                     TAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFED
                     GGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGE
                     IKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEG
                     RHSTGGMDELYK"
     primer_bind     complement(1003..1022)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1272..1290
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(1460..1483)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      1483..1492
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1506..1530)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(1527..1565)
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1688..1707)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1689..1707)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1714..1731)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1714..1730)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1723..1745)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1868..2170
                     /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     2049..2068
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2519..3313
                     /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             2519..3313
                     /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(2573..2592)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3183..3202
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      3513..3522
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3519..3893
                     /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      4031..4619
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4520..4539
                     /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 gcatcttaac
      721 cgcgggcctt gtacagctcg tccatgccgc cggtggagtg gcggccctcg gcgcgctcgt
      781 actgttccac gatggtgtag tcctcgttgt gggaggtgat gtccaacttg atgttgacgt
      841 tgtaggcgcc gggcagctgc acgggcttct tggccttgta ggtggtcttg acctcggcgt
      901 cgtagtggcc gccgtccttc agcttcagcc tctgcttgat ctcgcccttc agggcgccgt
      961 cctcggggta catccgctcg gaggaggcct cccagcccat cgtcttcttc tgcattacgg
     1021 ggccgtcgga ggggaagttg gtgccgcgca gcttcacctt gtagatgaac tcgccgtcct
     1081 gtagggagga gtcctgggtc acggtcacca cgccgccgtc ctcgaagttc atcacgcgct
     1141 cccacttgaa gccctcgggg aaggacagct tcaagtagtc ggggatgtcg gcggggtgct
     1201 tcacgtaggc cttggagccg tacatgaact gaggggacag gatgtcccag gcgaagggca
     1261 gggggccgcc cttggtcacc ttcagcttgg cggtctgggt gccctcgtag gggcggccct
     1321 cgccctcgcc ctcgatctcg aactcgtggc cgttcacgga gccctccatg tgcaccttga
     1381 agcgcatgaa ctccttgatg atggccatgt tatcctcctc gcccttgctc gcggtggaac
     1441 ccagtccgcc accggaagtt ttatcgtcgt catccttgta gtccatggtg gcgaattctc
     1501 caggcgatct gacggttcac taaacgagct ctgcttatat aggcctccca ccgtacacgc
     1561 ctacctcgcc ccactgtggg gtggagggga cactattacc ctgttatccc tacgtcgcaa
     1621 gggcgaattc tgcagatatc catcacactg gcggccgctc gagcatgcat ctagagggcc
     1681 caattcgccc tatagtgagt cgtattacaa ttcactggcc gtcgttttac aacgtcgtga
     1741 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     1801 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctata
     1861 cgtacggcag tttaaggttt acacctataa aagagagagc cgttatcgtc tgtttgtgga
     1921 tgtacagagt gatattattg acacgccggg gcgacggatg gtgatccccc tggccagtgc
     1981 acgtctgctg tcagataaag tctcccgtga actttacccg gtggtgcata tcggggatga
     2041 aagctggcgc atgatgacca ccgatatggc cagtgtgccg gtctccgtta tcggggaaga
     2101 agtggctgat ctcagccacc gcgaaaatga catcaaaaac gccattaacc tgatgttctg
     2161 gggaatataa atgtcaggca tgagattatc aaaaaggatc ttcacctaga tccttttcac
     2221 gtagaaagcc agtccgcaga aacggtgctg accccggatg aatgtcagct actgggctat
     2281 ctggacaagg gaaaacgcaa gcgcaaagag aaagcaggta gcttgcagtg ggcttacatg
     2341 gcgatagcta gactgggcgg ttttatggac agcaagcgaa ccggaattgc cagctggggc
     2401 gccctctggt aaggttggga agccctgcaa agtaaactgg atggctttct tgccgccaag
     2461 gatctgatgg cgcaggggat caagctctga tcaagagaca ggatgaggat cgtttcgcat
     2521 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     2581 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     2641 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     2701 agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     2761 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     2821 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     2881 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     2941 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     3001 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg
     3061 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     3121 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     3181 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     3241 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     3301 cgagttcttc tgaattatta acgcttacaa tttcctgatg cggtattttc tccttacgca
     3361 tctgtgcggt atttcacacc gcatcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     3421 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     3481 gataaatgct tcaataatag cacgtgagga gggccaccat ggccaagttg accagtgccg
     3541 ttccggtgct caccgcgcgc gacgtcgccg gagcggtcga gttctggacc gaccggctcg
     3601 ggttctcccg ggacttcgtg gaggacgact tcgccggtgt ggtccgggac gacgtgaccc
     3661 tgttcatcag cgcggtccag gaccaggtgg tgccggacaa caccctggcc tgggtgtggg
     3721 tgcgcggcct ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg aacttccggg
     3781 acgcctccgg gccggccatg accgagatcg gcgagcagcc gtgggggcgg gagttcgccc
     3841 tgcgcgaccc ggccggcaac tgcgtgcact tcgtggccga ggagcaggac tgacacgtgc
     3901 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     3961 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     4021 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     4081 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     4141 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     4201 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     4261 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     4321 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     4381 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     4441 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     4501 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     4561 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     4621 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     4681 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     4741 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
//
