LOCUS       sequence-148560-        4803 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Assays activity of transcriptional activators fused to SA Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4803)
  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 4803)
  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_SA-2x
FEATURES             Location/Qualifiers
     source          1..4803
                     /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(1690..1709)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1691..1709)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1716..1733)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1716..1732)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1725..1747)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1870..2172
                     /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     2051..2070
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2521..3315
                     /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             2521..3315
                     /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(2575..2594)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3185..3204
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      3516..3525
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3522..3896
                     /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      4034..4622
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4523..4542
                     /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 cctcgcacca ttcttcactg tggggtggag
     1561 gggactggta ctattaccct gttatcccta aagggcgaat tctgcagata tccatcacac
     1621 tggcggccga ttcttcactg tggggtggag gggactggtc tcgagcatgc atctagaggg
     1681 cccaattcgc cctatagtga gtcgtattac aattcactgg ccgtcgtttt acaacgtcgt
     1741 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     1801 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagccta
     1861 tacgtacggc agtttaaggt ttacacctat aaaagagaga gccgttatcg tctgtttgtg
     1921 gatgtacaga gtgatattat tgacacgccg gggcgacgga tggtgatccc cctggccagt
     1981 gcacgtctgc tgtcagataa agtctcccgt gaactttacc cggtggtgca tatcggggat
     2041 gaaagctggc gcatgatgac caccgatatg gccagtgtgc cggtctccgt tatcggggaa
     2101 gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc
     2161 tggggaatat aaatgtcagg catgagatta tcaaaaagga tcttcaccta gatccttttc
     2221 acgtagaaag ccagtccgca gaaacggtgc tgaccccgga tgaatgtcag ctactgggct
     2281 atctggacaa gggaaaacgc aagcgcaaag agaaagcagg tagcttgcag tgggcttaca
     2341 tggcgatagc tagactgggc ggttttatgg acagcaagcg aaccggaatt gccagctggg
     2401 gcgccctctg gtaaggttgg gaagccctgc aaagtaaact ggatggcttt ctcgccgcca
     2461 aggatctgat ggcgcagggg atcaagctct gatcaagaga caggatgagg atcgtttcgc
     2521 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     2581 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     2641 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     2701 caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     2761 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     2821 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     2881 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     2941 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     3001 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac
     3061 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     3121 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     3181 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     3241 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     3301 gacgagttct tctgaattat taacgcttac aatttcctga tgcggtattt tctccttacg
     3361 catctgtgcg gtatttcaca ccgcatacag gtggcacttt tcggggaaat gtgcgcggaa
     3421 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     3481 cctgataaat gcttcaataa tagcacgtga ggagggccac catggccaag ttgaccagtg
     3541 ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc
     3601 tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga
     3661 ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt
     3721 gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc
     3781 gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg
     3841 ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgacacg
     3901 tgctaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     3961 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     4021 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     4081 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     4141 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     4201 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     4261 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     4321 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     4381 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     4441 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     4501 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     4561 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     4621 aaaacgccag caacgcggcc tttttacggt tcctgggctt ttgctggcct tttgctcaca
     4681 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     4741 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     4801 aag
//
