LOCUS       sequence-140114-        4767 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TF reporter for gRNA-AAVS1_T1 (RNA-guided dTomato expression).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4767)
  AUTHORS   Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, Kosuri S, Yang
            L, Church GM
  TITLE     CAS9 transcriptional activators for target specificity screening and
            paired nickases for cooperative genome engineering.
  JOURNAL   Nat Biotechnol. 2013 Aug 1. doi: 10.1038/nbt.2675.
  PUBMED    23907171
REFERENCE   2  (bases 1 to 4767)
  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 
            Biotechnol. 2013 Aug 1. doi: 10.1038/nbt.2675."
COMMENT     Sequence Label: reporter-gT1
FEATURES             Location/Qualifiers
     source          1..4767
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     59..81
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     73..90
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     74..90
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     97..116
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        97..115
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        239..277
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     274..298
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      312..321
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             321..344
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             372..1070
                     /codon_start=1
                     /product="dimeric variant of DsRed fluorescent protein 
                     (Shaner et al., 2004)"
                     /label=dTomato
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGL
                     VTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIHQ
                     ALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRHH
                     LFLYGMDELYK"
     primer_bind     complement(502..520)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     770..789
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     964..987
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    1270..1351
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1307..1326)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        complement(1514..1532)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1515..1532)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1550..1566)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1550..1566)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1563..1585)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1574..1590
                     /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)."
     promoter        complement(join(1598..1604,1605..1622,1623..1628))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1643..1664
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(1952..2540)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2032..2051)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2678..3052)
                     /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"
     regulatory      3049..3058
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(3258..4052)
                     /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             complement(3258..4052)
                     /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(3369..3388)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3979..3998
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(4401..4703)
                     /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     complement(4503..4522)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
ORIGIN
        1 cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc
       61 cagtcacgac gttgtaaaac gacggccagt gaattgtaat acgactcact atagggcgaa
      121 ttgggccctc tagatgcatg ctcgagcggc cgccagtgtg atggatatct gcagaattcg
      181 cccttgcgac gtagggataa cagggtaata gtgtcccctc caccccacag tggggcgagg
      241 taggcgtgta cggtgggagg cctatataag cagagctcgt ttagtgaacc gtcagatcgc
      301 ctggagaatt cgccaccatg gactacaagg atgacgacga taaaacttcc ggtggcggac
      361 tgggttccac cgtgagcaag ggcgaggagg tcatcaaaga gttcatgcgc ttcaaggtgc
      421 gcatggaggg ctccatgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc
      481 cctacgaggg cacccagacc gccaagctga aggtgaccaa gggcggcccc ctgcccttcg
      541 cctgggacat cctgtccccc cagttcatgt acggctccaa ggcgtacgtg aagcaccccg
      601 ccgacatccc cgattacaag aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga
      661 tgaacttcga ggacggcggt ctggtgaccg tgacccagga ctcctccctg caggacggca
      721 cgctgatcta caaggtgaag atgcgcggca ccaacttccc ccccgacggc cccgtaatgc
      781 agaagaagac catgggctgg gaggcctcca ccgagcgcct gtacccccgc gacggcgtgc
      841 tgaagggcga gatccaccag gccctgaagc tgaaggacgg cggccactac ctggtggagt
      901 tcaagaccat ctacatggcc aagaagcccg tgcaactgcc cggctactac tacgtggaca
      961 ccaagctgga catcacctcc cacaacgagg actacaccat cgtggaacag tacgagcgct
     1021 ccgagggccg ccaccacctg ttcctgtacg gcatggacga gctgtacaag taagaattcg
     1081 agctcggtac ccggggatcc tctagtcagc tgacgcgtgc tagcgcggcc gcatcgataa
     1141 gcttgtcgac gatatctcta gaggatcata atcagccata ccacatttgt agaggtttta
     1201 cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt
     1261 gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     1321 aatttcacaa ataaagcatt tttttcactg cctcgagctt cctcgctcac tgactcgctg
     1381 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aataaagggc
     1441 gaattccagc acactggcgg ccgttactag tggatccgag ctcggtacca agcttgatgc
     1501 atagcttgag tattctatag tgtcacctaa atagcttggc gtaatcatgg tcatagctgt
     1561 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     1621 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     1681 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     1741 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     1801 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     1861 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     1921 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     1981 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     2041 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     2101 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     2161 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     2221 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     2281 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     2341 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     2401 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     2461 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     2521 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     2581 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     2641 agatcctttt aaattaaaaa tgaagtttta gcacgtgtca gtcctgctcc tcggccacga
     2701 agtgcacgca gttgccggcc gggtcgcgca gggcgaactc ccgcccccac ggctgctcgc
     2761 cgatctcggt catggccggc ccggaggcgt cccggaagtt cgtggacacg acctccgacc
     2821 actcggcgta cagctcgtcc aggccgcgca cccacaccca ggccagggtg ttgtccggca
     2881 ccacctggtc ctggaccgcg ctgatgaaca gggtcacgtc gtcccggacc acaccggcga
     2941 agtcgtcctc cacgaagtcc cgggagaacc cgagccggtc ggtccagaac tcgaccgctc
     3001 cggcgacgtc gcgcgcggtg agcaccggaa cggcactggt caacttggcc atggtggccc
     3061 tcctcacgtg ctattattga agcatttatc agggttattg tctcatgagc ggatacatat
     3121 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     3181 cacctgatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggaaa
     3241 ttgtaagcgt taataattca gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc
     3301 gaatcgggag cggcgatacc gtaaagcacg aggaagcggt cagcccattc gccgccaagc
     3361 tcttcagcaa tatcacgggt agccaacgct atgtcctgat agcggtccgc cacacccagc
     3421 cggccacagt cgatgaatcc agaaaagcgg ccattttcca ccatgatatt cggcaagcag
     3481 gcatcgccat gggtcacgac gagatcctcg ccgtcgggca tgctcgcctt gagcctggcg
     3541 aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg atcgacaaga
     3601 ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg
     3661 caggtagccg gatcaagcgt atgcagccgc cgcattgcat cagccatgat ggatactttc
     3721 tcggcaggag caaggtgaga tgacaggaga tcctgccccg gcacttcgcc caatagcagc
     3781 cagtcccttc ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg
     3841 gccagccacg atagccgcgc tgcctcgtct tgcagttcat tcagggcacc ggacaggtcg
     3901 gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc ggcatcagag
     3961 cagccgattg tctgttgtgc ccagtcatag ccgaatagcc tctccaccca agcggccgga
     4021 gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg atcctcatcc tgtctcttga
     4081 tcagagcttg atcccctgcg ccatcagatc cttggcggca agaaagccat ccagtttact
     4141 ttgcagggct tcccaacctt accagagggc gccccagctg gcaattccgg ttcgcttgct
     4201 gtccataaaa ccgcccagtc tagctatcgc catgtaagcc cactgcaagc tacctgcttt
     4261 ctctttgcgc ttgcgttttc ccttgtccag atagcccagt agctgacatt catccggggt
     4321 cagcaccgtt tctgcggact ggctttctac gtgaaaagga tctaggtgaa gatccttttt
     4381 gataatctca tgcctgacat ttatattccc cagaacatca ggttaatggc gtttttgatg
     4441 tcattttcgc ggtggctgag atcagccact tcttccccga taacggagac cggcacactg
     4501 gccatatcgg tggtcatcat gcgccagctt tcatccccga tatgcaccac cgggtaaagt
     4561 tcacgggaga ctttatctga cagcagacgt gcactggcca gggggatcac catccgtcgc
     4621 cccggcgtgt caataatatc actctgtaca tccacaaaca gacgataacg gctctctctt
     4681 ttataggtgt aaaccttaaa ctgccgtacg tataggctgc gcaactgttg ggaagggcga
     4741 tcggtgcggg cctcttcgct attacgc
//
