LOCUS       sequence-379983-        5561 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A plasmid encoding TRE3G sgRNA and TRE3G-promoter-Nluc.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5561)
  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 5561)
  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-Nluc
FEATURES             Location/Qualifiers
     source          1..5561
                     /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(696..1208)
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     CDS             complement(1236..1259)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      1259..1268
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(1285..1663)
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     primer_bind     complement(1352..1376)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    1421..1439
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1457..1475
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1493..1511
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1529..1547
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1565..1583
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1601..1619
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1637..1655
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_feature    1759..1876
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     misc_RNA        complement(1967..1985)
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        complement(2037..2055)
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     promoter        complement(2094..2342)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2152..2171)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2322..2342)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2449..2468)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2450..2468)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2475..2492)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2475..2491)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2484..2506)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             2629..2931
                     /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     2810..2829
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             3280..4074
                     /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             3280..4074
                     /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(3334..3353)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3944..3963
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      4274..4283
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4280..4654
                     /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      4792..5380
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5281..5300
                     /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 aattcttacg ccagaatgcg ttcgcacagc
      721 cgccagccgg tcactccgtt gatggttact cggaacagca gggagccgtc ggggttgatc
      781 aggcgctcgt cgataatttt gttgccgttc cacagggtcc ctgttacagt gatctttttg
      841 ccgtcgaaca cggcgatgcc ttcatacggc cgtccgaaat agtcgatcat gttcggcgta
      901 accccgtcga ttaccagtgt gccatagtgc aggatcacct taaagtgatg atcatccaca
      961 gggtacacca ccttaaaaat tttttcgatc tggcccattt ggtcgccgct cagaccttca
     1021 tacgggatga tgacatggat gtcgatcttc agcccatttt caccgctcag gacaatcctt
     1081 tggatcggag ttacggacac cccgagattc tgaaacaaac tggacacacc tccctgttca
     1141 aggacttggt ccaggttgta gccggctgtc tgtcgccagt ccccaacgaa atcttcgagt
     1201 gtgaagacgg tggaacccag tccgccaccg gaagttttat cgtcgtcatc cttgtagtcc
     1261 atggtggcgg gccccggtgt cgactttacg agggtaggaa gtggtacgga aagttggtat
     1321 aagacaaaag tgttgtggaa ttgctccagg cgatctgacg gttcactaaa cgagctctgc
     1381 ttttataggc gcccaccgta cacgcctaaa gcttatacgt tctctatcac tgatagggag
     1441 taaactggat atacgttctc tatcactgat agggagtaaa ctgtagatac gttctctatc
     1501 actgataggg agtaaactgg tcatacgttc tctatcactg atagggagta aactccttat
     1561 acgttctcta tcactgatag ggagtaaagt ctgcatacgt tctctatcac tgatagggag
     1621 taaactcttc atacgttctc tatcactgat agggagtaaa ctcgaggaat tcttgaagac
     1681 gaaagggcct cgtgagggcc caattcgccc tataggccgg cgccaaagtg gatctctgct
     1741 gtccctgtaa taaacccgaa aattttgaat ttttgtaatt tgtttttgta attctttagt
     1801 ttgtatgtct gttgctatta tgtctactat tctttcccct gcactgtacc ccccaatccc
     1861 cccttttctt ttaaaattgt ggatgaatac tgccatttgt ctcaagatct agttacgcca
     1921 agcggatcca aaaaaagcac cgactcggtg ccacttggcc ctgcagacat gggtgatcct
     1981 catgttggcc aagttgataa cggactagcc ttattttaac ttgctaggcc ctgcagacat
     2041 gggtgatcct catgttggcc tagctctaaa actatcagtg atagagaacg tacggtgttt
     2101 cgtcctttcc acaagatata taaagccaag aaatcgaaat actttcaagt tacggtaagc
     2161 atatgatagt ccattttaaa acataatttt aaaactgcaa actacccaag aaattattac
     2221 tttctacgtc acgtattttg tactaatatc tttgtgttta cagtcaaatt aattctaatt
     2281 atctctctaa cagccttgta tcgtatatgc aaatatgaag gaatcatggg aaataggccc
     2341 tctctgggtc ccctcggggt tgggaggtgg gtctgaaaca agggcgaatt ctgcagatat
     2401 ccatcacact ggcggccgct cgagcatgca tctagagggc ccaattcgcc ctatagtgag
     2461 tcgtattaca attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
     2521 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
     2581 gcccgcaccg atcgcccttc ccaacagttg cgcagcctat acgtacggca gtttaaggtt
     2641 tacacctata aaagagagag ccgttatcgt ctgtttgtgg atgtacagag tgatattatt
     2701 gacacgccgg ggcgacggat ggtgatcccc ctggccagtg cacgtctgct gtcagataaa
     2761 gtctcccgtg aactttaccc ggtggtgcat atcggggatg aaagctggcg catgatgacc
     2821 accgatatgg ccagtgtgcc ggtctccgtt atcggggaag aagtggctga tctcagccac
     2881 cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct ggggaatata aatgtcaggc
     2941 atgagattat caaaaaggat cttcacctag atccttttca cgtagaaagc cagtccgcag
     3001 aaacggtgct gaccccggat gaatgtcagc tactgggcta tctggacaag ggaaaacgca
     3061 agcgcaaaga gaaagcaggt agcttgcagt gggcttacat ggcgatagct agactgggcg
     3121 gttttatgga cagcaagcga accggaattg ccagctgggg cgccctctgg taaggttggg
     3181 aagccctgca aagtaaactg gatggctttc ttgccgccaa ggatctgatg gcgcagggga
     3241 tcaagctctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     3301 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     3361 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     3421 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta
     3481 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     3541 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     3601 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     3661 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     3721 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     3781 gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc
     3841 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     3901 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     3961 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     4021 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgaattatt
     4081 aacgcttaca atttcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     4141 cgcatcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     4201 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     4261 gcacgtgagg agggccacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg
     4321 cgacgtcgcc ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt
     4381 ggaggacgac ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca
     4441 ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct
     4501 gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat
     4561 gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa
     4621 ctgcgtgcac ttcgtggccg aggagcagga ctgacacgtg ctaaaacttc atttttaatt
     4681 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     4741 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     4801 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     4861 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     4921 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     4981 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     5041 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     5101 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     5161 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     5221 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     5281 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     5341 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     5401 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     5461 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     5521 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa g
//
