LOCUS       sequence-379977-        5316 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A plasmid encoding miniCMV sgRNA and miniCMV-promoter-Nluc.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5316)
  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 5316)
  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-Nluc
FEATURES             Location/Qualifiers
     source          1..5316
                     /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"
     primer_bind     complement(1282..1306)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(1303..1341)
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     misc_feature    1515..1632
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     misc_RNA        complement(1723..1741)
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        complement(1793..1811)
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     promoter        complement(1849..2097)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1907..1926)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2077..2097)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2204..2223)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2205..2223)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2230..2247)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2230..2246)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2239..2261)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             2384..2686
                     /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     2565..2584
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             3035..3829
                     /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             3035..3829
                     /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(3089..3108)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3699..3718
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      4029..4038
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4035..4409
                     /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      4547..5135
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5036..5055
                     /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 atggtggcga attctccagg cgatctgacg gttcactaaa cgagctctgc ttatataggc
     1321 ctcccaccgt acacgcctac ctcgccccac tgtggggtgg aggggacact attaccctgt
     1381 tatccctacg tcgcaagggc gaattctgca gatatccatc acactggcgg ccgctcgagc
     1441 atgcatctag agggcccaat tcgccctata ggccggcgcc aaagtggatc tctgctgtcc
     1501 ctgtaataaa cccgaaaatt ttgaattttt gtaatttgtt tttgtaattc tttagtttgt
     1561 atgtctgttg ctattatgtc tactattctt tcccctgcac tgtacccccc aatcccccct
     1621 tttcttttaa aattgtggat gaatactgcc atttgtctca agatctagtt acgccaagcg
     1681 gatccaaaaa aagcaccgac tcggtgccac ttggccctgc agacatgggt gatcctcatg
     1741 ttggccaagt tgataacgga ctagccttat tttaacttgc taggccctgc agacatgggt
     1801 gatcctcatg ttggcctagc tctaaaacca ctgtggggtg gaggggacgg tgtttcgtcc
     1861 tttccacaag atatataaag ccaagaaatc gaaatacttt caagttacgg taagcatatg
     1921 atagtccatt ttaaaacata attttaaaac tgcaaactac ccaagaaatt attactttct
     1981 acgtcacgta ttttgtacta atatctttgt gtttacagtc aaattaattc taattatctc
     2041 tctaacagcc ttgtatcgta tatgcaaata tgaaggaatc atgggaaata ggccctctct
     2101 gggtcccctc ggggttggga ggtgggtctg aaacaagggc gaattctgca gatatccatc
     2161 acactggcgg ccgctcgagc atgcatctag agggcccaat tcgccctata gtgagtcgta
     2221 ttacaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     2281 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     2341 caccgatcgc ccttcccaac agttgcgcag cctatacgta cggcagttta aggtttacac
     2401 ctataaaaga gagagccgtt atcgtctgtt tgtggatgta cagagtgata ttattgacac
     2461 gccggggcga cggatggtga tccccctggc cagtgcacgt ctgctgtcag ataaagtctc
     2521 ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc tggcgcatga tgaccaccga
     2581 tatggccagt gtgccggtct ccgttatcgg ggaagaagtg gctgatctca gccaccgcga
     2641 aaatgacatc aaaaacgcca ttaacctgat gttctgggga atataaatgt caggcatgag
     2701 attatcaaaa aggatcttca cctagatcct tttcacgtag aaagccagtc cgcagaaacg
     2761 gtgctgaccc cggatgaatg tcagctactg ggctatctgg acaagggaaa acgcaagcgc
     2821 aaagagaaag caggtagctt gcagtgggct tacatggcga tagctagact gggcggtttt
     2881 atggacagca agcgaaccgg aattgccagc tggggcgccc tctggtaagg ttgggaagcc
     2941 ctgcaaagta aactggatgg ctttcttgcc gccaaggatc tgatggcgca ggggatcaag
     3001 ctctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
     3061 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     3121 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     3181 caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg
     3241 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     3301 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     3361 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     3421 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     3481 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     3541 actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg
     3601 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     3661 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     3721 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     3781 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgaa ttattaacgc
     3841 ttacaatttc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     3901 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     3961 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatagcacg
     4021 tgaggagggc caccatggcc aagttgacca gtgccgttcc ggtgctcacc gcgcgcgacg
     4081 tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt ctcccgggac ttcgtggagg
     4141 acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg gtccaggacc
     4201 aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg cggcctggac gagctgtacg
     4261 ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc ctccgggccg gccatgaccg
     4321 agatcggcga gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc ggcaactgcg
     4381 tgcacttcgt ggccgaggag caggactgac acgtgctaaa acttcatttt taatttaaaa
     4441 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     4501 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     4561 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     4621 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     4681 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     4741 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     4801 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4861 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     4921 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     4981 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     5041 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     5101 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     5161 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     5221 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     5281 ccgagcgcag cgagtcagtg agcgaggaag cggaag
//
