LOCUS       sequence-192053-        4802 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Red fluorescent reporter that can be activated by dSa Cas9 activator
            indicating activity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4802)
  TITLE     Church Lab CRISPR plasmids 2017
REFERENCE   2  (bases 1 to 4802)
  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"
COMMENT     Sequence Label: CRISPR-Sa-TdTom. Reporter
FEATURES             Location/Qualifiers
     source          1..4802
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     24..43
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        24..42
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        201..239
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     236..260
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      274..283
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             283..306
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             334..1032
                     /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(464..482)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     732..751
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     926..949
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    1232..1313
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1269..1288)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        complement(1476..1494)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1477..1494)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1512..1528)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1512..1528)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1525..1547)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1536..1552
                     /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(1560..1566,1567..1584,1585..1590))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1605..1626
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(1914..2502)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1994..2013)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2640..3014)
                     /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      3011..3020
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(3220..4014)
                     /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(3220..4014)
                     /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(3331..3350)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3941..3960
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(4363..4665)
                     /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(4465..4484)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     join(4788..4802,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(4802,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtgaa ttgtaatacg actcactata gggcgaattg ggccctctag
       61 atgcatgctc gagaccagtc ccctccaccc cacagtgaag aatcggccgc cagtgtgatg
      121 gatatctgca gaattcgccc tttagggata acagggtaat agtaccagtc ccctccaccc
      181 cacagtgaag aatggtgcga ggtaggcgtg tacggtggga ggcctatata agcagagctc
      241 gtttagtgaa ccgtcagatc gcctggagaa ttcgccacca tggactacaa ggatgacgac
      301 gataaaactt ccggtggcgg actgggttcc accgtgagca agggcgagga ggtcatcaaa
      361 gagttcatgc gcttcaaggt gcgcatggag ggctccatga acggccacga gttcgagatc
      421 gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc
      481 aagggcggcc ccctgccctt cgcctgggac atcctgtccc cccagttcat gtacggctcc
      541 aaggcgtacg tgaagcaccc cgccgacatc cccgattaca agaagctgtc cttccccgag
      601 ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gtctggtgac cgtgacccag
      661 gactcctccc tgcaggacgg cacgctgatc tacaaggtga agatgcgcgg caccaacttc
      721 ccccccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc caccgagcgc
      781 ctgtaccccc gcgacggcgt gctgaagggc gagatccacc aggccctgaa gctgaaggac
      841 ggcggccact acctggtgga gttcaagacc atctacatgg ccaagaagcc cgtgcaactg
      901 cccggctact actacgtgga caccaagctg gacatcacct cccacaacga ggactacacc
      961 atcgtggaac agtacgagcg ctccgagggc cgccaccacc tgttcctgta cggcatggac
     1021 gagctgtaca agtaagaatt cgagctcggt acccggggat cctctagtca gctgacgcgt
     1081 gctagcgcgg ccgcatcgat aagcttgtcg acgatatctc tagaggatca taatcagcca
     1141 taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct
     1201 gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta
     1261 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcctcgagc
     1321 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     1381 ctcaaaggcg gtaataaagg gcgaattcca gcacactggc ggccgttact agtggatccg
     1441 agctcggtac caagcttgat gcatagcttg agtattctat agtgtcacct aaatagcttg
     1501 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     1561 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     1621 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     1681 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     1741 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     1801 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     1861 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     1921 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     1981 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     2041 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     2101 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     2161 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     2221 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     2281 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     2341 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     2401 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     2461 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     2521 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     2581 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt tagcacgtgt
     2641 cagtcctgct cctcggccac gaagtgcacg cagttgccgg ccgggtcgcg cagggcgaac
     2701 tcccgccccc acggctgctc gccgatctcg gtcatggccg gcccggaggc gtcccggaag
     2761 ttcgtggaca cgacctccga ccactcggcg tacagctcgt ccaggccgcg cacccacacc
     2821 caggccaggg tgttgtccgg caccacctgg tcctggaccg cgctgatgaa cagggtcacg
     2881 tcgtcccgga ccacaccggc gaagtcgtcc tccacgaagt cccgggagaa cccgagccgg
     2941 tcggtccaga actcgaccgc tccggcgacg tcgcgcgcgg tgagcaccgg aacggcactg
     3001 gtcaacttgg ccatggtggc cctcctcacg tgctattatt gaagcattta tcagggttat
     3061 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     3121 cgcacatttc cccgaaaagt gccacctgat gcggtgtgaa ataccgcaca gatgcgtaag
     3181 gagaaaatac cgcatcagga aattgtaagc gttaataatt cagaagaact cgtcaagaag
     3241 gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg
     3301 gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg ctatgtcctg
     3361 atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc ggccattttc
     3421 caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct cgccgtcggg
     3481 catgctcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat gctcttcgtc
     3541 cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct cgatgcgatg
     3601 tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc
     3661 atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga gatcctgccc
     3721 cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt cgagcacagc
     3781 tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt cttgcagttc
     3841 attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct gcgctgacag
     3901 ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat agccgaatag
     3961 cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa tcatgcgaaa
     4021 cgatcctcat cctgtctctt gatcagagct tgatcccctg cgccatcaga tccttggcgg
     4081 caagaaagcc atccagttta ctttgcaggg cttcccaacc ttaccagagg gcgccccagc
     4141 tggcaattcc ggttcgcttg ctgtccataa aaccgcccag tctagctatc gccatgtaag
     4201 cccactgcaa gctacctgct ttctctttgc gcttgcgttt tcccttgtcc agatagccca
     4261 gtagctgaca ttcatccggg gtcagcaccg tttctgcgga ctggctttct acgtgaaaag
     4321 gatctaggtg aagatccttt ttgataatct catgcctgac atttatattc cccagaacat
     4381 caggttaatg gcgtttttga tgtcattttc gcggtggctg agatcagcca cttcttcccc
     4441 gataacggag accggcacac tggccatatc ggtggtcatc atgcgccagc tttcatcccc
     4501 gatatgcacc accgggtaaa gttcacggga gactttatct gacagcagac gtgcactggc
     4561 cagggggatc accatccgtc gccccggcgt gtcaataata tcactctgta catccacaaa
     4621 cagacgataa cggctctctc ttttataggt gtaaacctta aactgccgta cgtataggct
     4681 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
     4741 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
     4801 tt
//
