LOCUS       sequence-148561-        4791 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Assays activity of transcriptional activators fused to SP Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4791)
  AUTHORS   Kiani S, Chavez A, Tuttle M, Hall RN, Chari R, Ter-Ovanesyan D, Qian
            J, Pruitt BW, Beal J, Vora S, Buchthal J, Kowal EJ, Ebrahimkhani MR,
            Collins JJ, Weiss R, Church G
  TITLE     Cas9 gRNA engineering for genome editing, activation and repression.
  JOURNAL   Nat Methods. 2015 Sep 7. doi: 10.1038/nmeth.3580.
  PUBMED    26344044
REFERENCE   2  (bases 1 to 4791)
  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 
            Methods. 2015 Sep 7. doi: 10.1038/nmeth.3580."
COMMENT     Sequence Label: gRNA_tdtomato_reporter1_SP-2x
FEATURES             Location/Qualifiers
     source          1..4791
                     /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(701..1399)
                     /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(784..807)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(982..1001)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1251..1269
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(1427..1450)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      1450..1459
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1473..1497)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(1494..1532)
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1704..1721)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1704..1720)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1713..1735)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1858..2160
                     /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     2039..2058
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2509..3303
                     /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             2509..3303
                     /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(2563..2582)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3173..3192
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      3504..3513
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3510..3884
                     /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      4022..4610
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4511..4530
                     /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 cgaattctta cttgtacagc tcgtccatgc
      721 cgtacaggaa caggtggtgg cggccctcgg agcgctcgta ctgttccacg atggtgtagt
      781 cctcgttgtg ggaggtgatg tccagcttgg tgtccacgta gtagtagccg ggcagttgca
      841 cgggcttctt ggccatgtag atggtcttga actccaccag gtagtggccg ccgtccttca
      901 gcttcagggc ctggtggatc tcgcccttca gcacgccgtc gcgggggtac aggcgctcgg
      961 tggaggcctc ccagcccatg gtcttcttct gcattacggg gccgtcgggg gggaagttgg
     1021 tgccgcgcat cttcaccttg tagatcagcg tgccgtcctg cagggaggag tcctgggtca
     1081 cggtcaccag accgccgtcc tcgaagttca tcacgcgctc ccacttgaag ccctcgggga
     1141 aggacagctt cttgtaatcg gggatgtcgg cggggtgctt cacgtacgcc ttggagccgt
     1201 acatgaactg gggggacagg atgtcccagg cgaagggcag ggggccgccc ttggtcacct
     1261 tcagcttggc ggtctgggtg ccctcgtagg ggcggccctc gccctcgccc tcgatctcga
     1321 actcgtggcc gttcatggag ccctccatgc gcaccttgaa gcgcatgaac tctttgatga
     1381 cctcctcgcc cttgctcacg gtggaaccca gtccgccacc ggaagtttta tcgtcgtcat
     1441 ccttgtagtc catggtggcg aattctccag gcgatctgac ggttcactaa acgagctctg
     1501 cttatatagg cctcccaccg tacacgccta cctcgcccca ctgtggggtg gaggggacac
     1561 tattaccctg ttatccctac gtcgcaaggg cgaattctgc agatatccat cacactggcg
     1621 gccgctcgag catgcatcta gagggcccaa ttcgccctat agtgccccac tgtggggtgg
     1681 aggggacagt cgtattacaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     1741 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     1801 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctata cgtacggcag
     1861 tttaaggttt acacctataa aagagagagc cgttatcgtc tgtttgtgga tgtacagagt
     1921 gatattattg acacgccggg gcgacggatg gtgatccccc tggccagtgc acgtctgctg
     1981 tcagataaag tctcccgtga actttacccg gtggtgcata tcggggatga aagctggcgc
     2041 atgatgacca ccgatatggc cagtgtgccg gtctccgtta tcggggaaga agtggctgat
     2101 ctcagccacc gcgaaaatga catcaaaaac gccattaacc tgatgttctg gggaatataa
     2161 atgtcaggca tgagattatc aaaaaggatc ttcacctaga tccttttcac gtagaaagcc
     2221 agtccgcaga aacggtgctg accccggatg aatgtcagct actgggctat ctggacaagg
     2281 gaaaacgcaa gcgcaaagag aaagcaggta gcttgcagtg ggcttacatg gcgatagcta
     2341 gactgggcgg ttttatggac agcaagcgaa ccggaattgc cagctggggc gccctctggt
     2401 aaggttggga agccctgcaa agtaaactgg atggctttct cgccgccaag gatctgatgg
     2461 cgcaggggat caagctctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa
     2521 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     2581 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     2641 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca
     2701 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     2761 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     2821 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     2881 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     2941 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     3001 ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc
     3061 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     3121 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     3181 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     3241 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     3301 tgaattatta acgcttacaa tttcctgatg cggtattttc tccttacgca tctgtgcggt
     3361 atttcacacc gcatacaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     3421 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     3481 ttcaataata gcacgtgagg agggccacca tggccaagtt gaccagtgcc gttccggtgc
     3541 tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac cgaccggctc gggttctccc
     3601 gggacttcgt ggaggacgac ttcgccggtg tggtccggga cgacgtgacc ctgttcatca
     3661 gcgcggtcca ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc
     3721 tggacgagct gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg
     3781 ggccggccat gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc
     3841 cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga ctgacacgtg ctaaaacttc
     3901 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     3961 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     4021 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     4081 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     4141 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     4201 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     4261 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     4321 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     4381 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     4441 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     4501 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     4561 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     4621 acgcggcctt tttacggttc ctgggctttt gctggccttt tgctcacatg ttctttcctg
     4681 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     4741 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa g
//
