LOCUS       sequence-379894-        5505 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A plasmid encoding miniCMV sgRNA and miniCMV-promoter-TdTomato.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5505)
  AUTHORS   Omachi K, Miner JH
  TITLE     Comparative analysis of dCas9-VP64 variants and multiplexed guide 
            RNAs mediating CRISPR activation.
  JOURNAL   PLoS One. 2022 Jun 28;17(6):e0270008. doi: 
            10.1371/journal.pone.0270008. eCollection 2022.
  PUBMED    35763517
REFERENCE   2  (bases 1 to 5505)
  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: "PLoS One.";
            date: "2022-06-28"; volume: "17(6):e0270008. doi"; pages: " 
            10.1371/journal.pone.0270008. eCollection 2022"
COMMENT     Sequence Label: pCR-U6-gRNA-miniCMV-TdTomato
FEATURES             Location/Qualifiers
     source          1..5505
                     /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"
     promoter        374..622
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     374..394
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     545..564
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        660..678
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        730..748
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_feature    839..956
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1130..1168
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1165..1189
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1203..1212
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1212..1235
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1263..1961
                     /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(1393..1411)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1661..1680
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1855..1878
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    2161..2242
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2198..2217)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2393..2412)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2394..2412)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2419..2436)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2419..2435)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2428..2450)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             2573..2875
                     /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     2754..2773
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             3224..4018
                     /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             3224..4018
                     /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(3278..3297)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3888..3907
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      4218..4227
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4224..4598
                     /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      4736..5324
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5225..5244
                     /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 gcccttgttt cagacccacc tcccaacccc
      361 gaggggaccc agagagggcc tatttcccat gattccttca tatttgcata tacgatacaa
      421 ggctgttaga gagataatta gaattaattt gactgtaaac acaaagatat tagtacaaaa
      481 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
      541 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
      601 cttgtggaaa ggacgaaaca ccgtcccctc caccccacag tggttttaga gctaggccaa
      661 catgaggatc acccatgtct gcagggccta gcaagttaaa ataaggctag tccgttatca
      721 acttggccaa catgaggatc acccatgtct gcagggccaa gtggcaccga gtcggtgctt
      781 tttttggatc cgcttggcgt aactagatct tgagacaaat ggcagtattc atccacaatt
      841 ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag
      901 caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg
      961 tttattacag ggacagcaga gatccacttt ggcgccggcc tatagggcga attgggccct
     1021 ctagatgcat gctcgagcgg ccgccagtgt gatggatatc tgcagaattc gcccttgcga
     1081 cgtagggata acagggtaat agtgtcccct ccaccccaca gtggggcgag gtaggcgtgt
     1141 acggtgggag gcctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagaat
     1201 tcgccaccat ggactacaag gatgacgacg ataaaacttc cggtggcgga ctgggttcca
     1261 ccgtgagcaa gggcgaggag gtcatcaaag agttcatgcg cttcaaggtg cgcatggagg
     1321 gctccatgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg
     1381 gcacccagac cgccaagctg aaggtgacca agggcggccc cctgcccttc gcctgggaca
     1441 tcctgtcccc ccagttcatg tacggctcca aggcgtacgt gaagcacccc gccgacatcc
     1501 ccgattacaa gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg
     1561 aggacggcgg tctggtgacc gtgacccagg actcctccct gcaggacggc acgctgatct
     1621 acaaggtgaa gatgcgcggc accaacttcc cccccgacgg ccccgtaatg cagaagaaga
     1681 ccatgggctg ggaggcctcc accgagcgcc tgtacccccg cgacggcgtg ctgaagggcg
     1741 agatccacca ggccctgaag ctgaaggacg gcggccacta cctggtggag ttcaagacca
     1801 tctacatggc caagaagccc gtgcaactgc ccggctacta ctacgtggac accaagctgg
     1861 acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgagcgc tccgagggcc
     1921 gccaccacct gttcctgtac ggcatggacg agctgtacaa gtaagaattc gagctcggta
     1981 cccggggatc ctctagtcag ctgacgcgtg ctagcgcggc cgcatcgata agcttgtcga
     2041 cgatatctct agaggatcat aatcagccat accacatttg tagaggtttt acttgcttta
     2101 aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt
     2161 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     2221 aataaagcat ttttttcact gcctcgagct tcctcgctca ctgactcgct gcagagagga
     2281 atctttgcag ctaatggacc ttctaggtct tgaaaggagt gggaagggcg aattctgcag
     2341 atatccatca cactggcggc cgctcgagca tgcatctaga gggcccaatt cgccctatag
     2401 tgagtcgtat tacaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
     2461 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
     2521 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctatacgtac ggcagtttaa
     2581 ggtttacacc tataaaagag agagccgtta tcgtctgttt gtggatgtac agagtgatat
     2641 tattgacacg ccggggcgac ggatggtgat ccccctggcc agtgcacgtc tgctgtcaga
     2701 taaagtctcc cgtgaacttt acccggtggt gcatatcggg gatgaaagct ggcgcatgat
     2761 gaccaccgat atggccagtg tgccggtctc cgttatcggg gaagaagtgg ctgatctcag
     2821 ccaccgcgaa aatgacatca aaaacgccat taacctgatg ttctggggaa tataaatgtc
     2881 aggcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttcacgtaga aagccagtcc
     2941 gcagaaacgg tgctgacccc ggatgaatgt cagctactgg gctatctgga caagggaaaa
     3001 cgcaagcgca aagagaaagc aggtagcttg cagtgggctt acatggcgat agctagactg
     3061 ggcggtttta tggacagcaa gcgaaccgga attgccagct ggggcgccct ctggtaaggt
     3121 tgggaagccc tgcaaagtaa actggatggc tttcttgccg ccaaggatct gatggcgcag
     3181 gggatcaagc tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg
     3241 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
     3301 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
     3361 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg
     3421 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
     3481 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
     3541 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
     3601 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
     3661 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     3721 gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt
     3781 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     3841 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     3901 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     3961 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgaat
     4021 tattaacgct tacaatttcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     4081 acaccgcatc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     4141 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     4201 aatagcacgt gaggagggcc accatggcca agttgaccag tgccgttccg gtgctcaccg
     4261 cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg gctcgggttc tcccgggact
     4321 tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt gaccctgttc atcagcgcgg
     4381 tccaggacca ggtggtgccg gacaacaccc tggcctgggt gtgggtgcgc ggcctggacg
     4441 agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt ccgggacgcc tccgggccgg
     4501 ccatgaccga gatcggcgag cagccgtggg ggcgggagtt cgccctgcgc gacccggccg
     4561 gcaactgcgt gcacttcgtg gccgaggagc aggactgaca cgtgctaaaa cttcattttt
     4621 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     4681 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     4741 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     4801 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     4861 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     4921 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     4981 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     5041 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     5101 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     5161 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     5221 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     5281 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     5341 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     5401 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     5461 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaag
//
