LOCUS       sequence-379981-        5749 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A plasmid encoding TRE3G sgRNA and TRE3G-promoter-TdTomato.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5749)
  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 5749)
  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-TRE3G-TdTomato
FEATURES             Location/Qualifiers
     source          1..5749
                     /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    418..499
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     443..462
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(699..1397)
                     /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(782..805)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(980..999)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1249..1267
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(1425..1448)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(1473..1851)
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     primer_bind     complement(1540..1564)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    1609..1627
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1645..1663
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1681..1699
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1717..1735
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1753..1771
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1789..1807
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1825..1843
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_feature    1947..2064
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     misc_RNA        complement(2155..2173)
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        complement(2225..2243)
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     promoter        complement(2282..2530)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2340..2359)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2510..2530)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2637..2656)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2638..2656)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2663..2680)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2663..2679)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2672..2694)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             2817..3119
                     /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     2998..3017
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             3468..4262
                     /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             3468..4262
                     /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(3522..3541)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4132..4151
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      4462..4471
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4468..4842
                     /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      4980..5568
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5469..5488
                     /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 gcccttccca ctcctttcaa gacctagaag
      361 gtccattagc tgcaaagatt cctctctgca gcgagtcagt gagcgaggaa gctcgaggca
      421 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat
      481 aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg
      541 ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta tggctgatta
      601 tgatcctcta gagatatcgt cgacaagctt atcgatgcgg ccgcgctagc acgcgtcagc
      661 tgactagagg atccccgggt accgagctcg aattcttact tgtacagctc gtccatgccg
      721 tacaggaaca ggtggtggcg gccctcggag cgctcgtact gttccacgat ggtgtagtcc
      781 tcgttgtggg aggtgatgtc cagcttggtg tccacgtagt agtagccggg cagttgcacg
      841 ggcttcttgg ccatgtagat ggtcttgaac tccaccaggt agtggccgcc gtccttcagc
      901 ttcagggcct ggtggatctc gcccttcagc acgccgtcgc gggggtacag gcgctcggtg
      961 gaggcctccc agcccatggt cttcttctgc attacggggc cgtcgggggg gaagttggtg
     1021 ccgcgcatct tcaccttgta gatcagcgtg ccgtcctgca gggaggagtc ctgggtcacg
     1081 gtcaccagac cgccgtcctc gaagttcatc acgcgctccc acttgaagcc ctcggggaag
     1141 gacagcttct tgtaatcggg gatgtcggcg gggtgcttca cgtacgcctt ggagccgtac
     1201 atgaactggg gggacaggat gtcccaggcg aagggcaggg ggccgccctt ggtcaccttc
     1261 agcttggcgg tctgggtgcc ctcgtagggg cggccctcgc cctcgccctc gatctcgaac
     1321 tcgtggccgt tcatggagcc ctccatgcgc accttgaagc gcatgaactc tttgatgacc
     1381 tcctcgccct tgctcacggt ggaacccagt ccgccaccgg aagttttatc gtcgtcatcc
     1441 ttgtagtcca tggtgcgggc cccggtgtcg actttacgag ggtaggaagt ggtacggaaa
     1501 gttggtataa gacaaaagtg ttgtggaatt gctccaggcg atctgacggt tcactaaacg
     1561 agctctgctt ttataggcgc ccaccgtaca cgcctaaagc ttatacgttc tctatcactg
     1621 atagggagta aactggatat acgttctcta tcactgatag ggagtaaact gtagatacgt
     1681 tctctatcac tgatagggag taaactggtc atacgttctc tatcactgat agggagtaaa
     1741 ctccttatac gttctctatc actgataggg agtaaagtct gcatacgttc tctatcactg
     1801 atagggagta aactcttcat acgttctcta tcactgatag ggagtaaact cgaggaattc
     1861 ttgaagacga aagggcctcg tgagggccca attcgcccta taggccggcg ccaaagtgga
     1921 tctctgctgt ccctgtaata aacccgaaaa ttttgaattt ttgtaatttg tttttgtaat
     1981 tctttagttt gtatgtctgt tgctattatg tctactattc tttcccctgc actgtacccc
     2041 ccaatccccc cttttctttt aaaattgtgg atgaatactg ccatttgtct caagatctag
     2101 ttacgccaag cggatccaaa aaaagcaccg actcggtgcc acttggccct gcagacatgg
     2161 gtgatcctca tgttggccaa gttgataacg gactagcctt attttaactt gctaggccct
     2221 gcagacatgg gtgatcctca tgttggccta gctctaaaac tatcagtgat agagaacgta
     2281 cggtgtttcg tcctttccac aagatatata aagccaagaa atcgaaatac tttcaagtta
     2341 cggtaagcat atgatagtcc attttaaaac ataattttaa aactgcaaac tacccaagaa
     2401 attattactt tctacgtcac gtattttgta ctaatatctt tgtgtttaca gtcaaattaa
     2461 ttctaattat ctctctaaca gccttgtatc gtatatgcaa atatgaagga atcatgggaa
     2521 ataggccctc tctgggtccc ctcggggttg ggaggtgggt ctgaaacaag ggcgaattct
     2581 gcagatatcc atcacactgg cggccgctcg agcatgcatc tagagggccc aattcgccct
     2641 atagtgagtc gtattacaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     2701 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     2761 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctatac gtacggcagt
     2821 ttaaggttta cacctataaa agagagagcc gttatcgtct gtttgtggat gtacagagtg
     2881 atattattga cacgccgggg cgacggatgg tgatccccct ggccagtgca cgtctgctgt
     2941 cagataaagt ctcccgtgaa ctttacccgg tggtgcatat cggggatgaa agctggcgca
     3001 tgatgaccac cgatatggcc agtgtgccgg tctccgttat cggggaagaa gtggctgatc
     3061 tcagccaccg cgaaaatgac atcaaaaacg ccattaacct gatgttctgg ggaatataaa
     3121 tgtcaggcat gagattatca aaaaggatct tcacctagat ccttttcacg tagaaagcca
     3181 gtccgcagaa acggtgctga ccccggatga atgtcagcta ctgggctatc tggacaaggg
     3241 aaaacgcaag cgcaaagaga aagcaggtag cttgcagtgg gcttacatgg cgatagctag
     3301 actgggcggt tttatggaca gcaagcgaac cggaattgcc agctggggcg ccctctggta
     3361 aggttgggaa gccctgcaaa gtaaactgga tggctttctt gccgccaagg atctgatggc
     3421 gcaggggatc aagctctgat caagagacag gatgaggatc gtttcgcatg attgaacaag
     3481 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     3541 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     3601 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag
     3661 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     3721 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     3781 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     3841 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     3901 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     3961 tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg
     4021 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     4081 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     4141 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     4201 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     4261 gaattattaa cgcttacaat ttcctgatgc ggtattttct ccttacgcat ctgtgcggta
     4321 tttcacaccg catcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     4381 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     4441 caataatagc acgtgaggag ggccaccatg gccaagttga ccagtgccgt tccggtgctc
     4501 accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg accggctcgg gttctcccgg
     4561 gacttcgtgg aggacgactt cgccggtgtg gtccgggacg acgtgaccct gttcatcagc
     4621 gcggtccagg accaggtggt gccggacaac accctggcct gggtgtgggt gcgcggcctg
     4681 gacgagctgt acgccgagtg gtcggaggtc gtgtccacga acttccggga cgcctccggg
     4741 ccggccatga ccgagatcgg cgagcagccg tgggggcggg agttcgccct gcgcgacccg
     4801 gccggcaact gcgtgcactt cgtggccgag gagcaggact gacacgtgct aaaacttcat
     4861 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     4921 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     4981 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     5041 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     5101 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     5161 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     5221 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     5281 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     5341 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     5401 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     5461 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     5521 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     5581 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     5641 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     5701 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaag
//
