LOCUS       sequence-148559-        4799 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Assays activity of transcriptional activators fused to ST1 Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4799)
  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 4799)
  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_reporter_1_ST1_2x_binding
FEATURES             Location/Qualifiers
     source          1..4799
                     /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(1686..1705)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1687..1705)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1712..1729)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1712..1728)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1721..1743)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1866..2168
                     /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     2047..2066
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2517..3311
                     /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             2517..3311
                     /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(2571..2590)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3181..3200
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      3512..3521
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3518..3892
                     /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      4030..4618
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4519..4538
                     /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 cctcgtttct tgcactgtgg ggtggagggg
     1561 acactattac cctgttatcc ctacgtcgca agggcgaatt ctgcagatat ccatcacact
     1621 ggcggccgtt tcttgcactg tggggtggag gggacctcga gcatgcatct agagggccca
     1681 attcgcccta tagtgagtcg tattacaatt cactggccgt cgttttacaa cgtcgtgact
     1741 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     1801 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctatacg
     1861 tacggcagtt taaggtttac acctataaaa gagagagccg ttatcgtctg tttgtggatg
     1921 tacagagtga tattattgac acgccggggc gacggatggt gatccccctg gccagtgcac
     1981 gtctgctgtc agataaagtc tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa
     2041 gctggcgcat gatgaccacc gatatggcca gtgtgccggt ctccgttatc ggggaagaag
     2101 tggctgatct cagccaccgc gaaaatgaca tcaaaaacgc cattaacctg atgttctggg
     2161 gaatataaat gtcaggcatg agattatcaa aaaggatctt cacctagatc cttttcacgt
     2221 agaaagccag tccgcagaaa cggtgctgac cccggatgaa tgtcagctac tgggctatct
     2281 ggacaaggga aaacgcaagc gcaaagagaa agcaggtagc ttgcagtggg cttacatggc
     2341 gatagctaga ctgggcggtt ttatggacag caagcgaacc ggaattgcca gctggggcgc
     2401 cctctggtaa ggttgggaag ccctgcaaag taaactggat ggctttctcg ccgccaagga
     2461 tctgatggcg caggggatca agctctgatc aagagacagg atgaggatcg tttcgcatga
     2521 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     2581 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     2641 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag
     2701 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     2761 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     2821 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     2881 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     2941 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     3001 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg
     3061 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     3121 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     3181 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     3241 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     3301 agttcttctg aattattaac gcttacaatt tcctgatgcg gtattttctc cttacgcatc
     3361 tgtgcggtat ttcacaccgc atacaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     3421 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     3481 ataaatgctt caataatagc acgtgaggag ggccaccatg gccaagttga ccagtgccgt
     3541 tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg accggctcgg
     3601 gttctcccgg gacttcgtgg aggacgactt cgccggtgtg gtccgggacg acgtgaccct
     3661 gttcatcagc gcggtccagg accaggtggt gccggacaac accctggcct gggtgtgggt
     3721 gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc gtgtccacga acttccggga
     3781 cgcctccggg ccggccatga ccgagatcgg cgagcagccg tgggggcggg agttcgccct
     3841 gcgcgacccg gccggcaact gcgtgcactt cgtggccgag gagcaggact gacacgtgct
     3901 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     3961 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     4021 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     4081 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     4141 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     4201 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     4261 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     4321 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     4381 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     4441 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     4501 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     4561 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     4621 cgccagcaac gcggcctttt tacggttcct gggcttttgc tggccttttg ctcacatgtt
     4681 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     4741 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaag
//
