LOCUS       sequence-225157-        5315 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pPN10 with gRNA for spCRISPR-Cas9 targeted to CTG repeat,  PAM: CTG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5315)
  AUTHORS   Cinesi C, Aeschbach L, Yang B, Dion V
  TITLE     Contracting CAG/CTG repeats using the CRISPR-Cas9 nickase.
  JOURNAL   Nat Commun. 2016 Nov 9;7:13272. doi: 10.1038/ncomms13272.
  PUBMED    27827362
REFERENCE   2  (bases 1 to 5315)
  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: "Nat 
            Commun."; date: "2016-11-9"; volume: "7:13272. doi"; pages: " 
            10.1038/ncomms13272"
COMMENT     Sequence Label: pPN10-gCTG
FEATURES             Location/Qualifiers
     source          1..5315
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        20..268
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     20..40
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     191..210
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        289..364
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(376..400)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(485..503)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(486..503)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(521..537)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(521..537)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(534..556)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    545..561
                     /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(569..575,576..593,594..599))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    614..635
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(752..769)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(923..1511)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1003..1022)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1649..2023)
                     /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      2020..2029
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2229..3023)
                     /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(2229..3023)
                     /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(2340..2359)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2950..2969
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(3372..3674)
                     /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(3474..3493)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     3797..3819
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3811..3828
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3812..3828
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3835..3854
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3835..3853
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    3885..3966
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     3910..3929
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4196..4795)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4279..4299)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     4776..4795
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(4892..5221)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4906..5041
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 taccaaggtc gggcaggaag agggcctatt tcccatgatt ccttcatatt tgcatatacg
       61 atacaaggct gttagagaga taattagaat taatttgact gtaaacacaa agatattagt
      121 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
      181 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt
      241 atatatcttg tggaaaggac gaaacaccgc tgctgctgct gctgctgcgt tttagagcta
      301 gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
      361 gtgctttttt tctagaccca gctttcttgt acaaagttgg cattaaaggg cgaattccag
      421 cacactggcg gccgttacta gtggatccga gctcggtacc aagcttgatg catagcttga
      481 gtattctata gtgtcaccta aatagcttgg cgtaatcatg gtcatagctg tttcctgtgt
      541 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
      601 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
      661 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
      721 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
      781 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
      841 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
      901 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
      961 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     1021 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     1081 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     1141 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     1201 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     1261 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     1321 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     1381 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     1441 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     1501 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     1561 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     1621 taaattaaaa atgaagtttt agcacgtgtc agtcctgctc ctcggccacg aagtgcacgc
     1681 agttgccggc cgggtcgcgc agggcgaact cccgccccca cggctgctcg ccgatctcgg
     1741 tcatggccgg cccggaggcg tcccggaagt tcgtggacac gacctccgac cactcggcgt
     1801 acagctcgtc caggccgcgc acccacaccc aggccagggt gttgtccggc accacctggt
     1861 cctggaccgc gctgatgaac agggtcacgt cgtcccggac cacaccggcg aagtcgtcct
     1921 ccacgaagtc ccgggagaac ccgagccggt cggtccagaa ctcgaccgct ccggcgacgt
     1981 cgcgcgcggt gagcaccgga acggcactgg tcaacttggc catggtggcc ctcctcacgt
     2041 gctattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     2101 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgatg
     2161 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggaa attgtaagcg
     2221 ttaataattc agaagaactc gtcaagaagg cgatagaagg cgatgcgctg cgaatcggga
     2281 gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag ctcttcagca
     2341 atatcacggg tagccaacgc tatgtcctga tagcggtccg ccacacccag ccggccacag
     2401 tcgatgaatc cagaaaagcg gccattttcc accatgatat tcggcaagca ggcatcgcca
     2461 tgggtcacga cgagatcctc gccgtcgggc atgctcgcct tgagcctggc gaacagttcg
     2521 gctggcgcga gcccctgatg ctcttcgtcc agatcatcct gatcgacaag accggcttcc
     2581 atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg gcaggtagcc
     2641 ggatcaagcg tatgcagccg ccgcattgca tcagccatga tggatacttt ctcggcagga
     2701 gcaaggtgag atgacaggag atcctgcccc ggcacttcgc ccaatagcag ccagtccctt
     2761 cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt ggccagccac
     2821 gatagccgcg ctgcctcgtc ttgcagttca ttcagggcac cggacaggtc ggtcttgaca
     2881 aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg cggcatcaga gcagccgatt
     2941 gtctgttgtg cccagtcata gccgaatagc ctctccaccc aagcggccgg agaacctgcg
     3001 tgcaatccat cttgttcaat catgcgaaac gatcctcatc ctgtctcttg atcagagctt
     3061 gatcccctgc gccatcagat ccttggcggc aagaaagcca tccagtttac tttgcagggc
     3121 ttcccaacct taccagaggg cgccccagct ggcaattccg gttcgcttgc tgtccataaa
     3181 accgcccagt ctagctatcg ccatgtaagc ccactgcaag ctacctgctt tctctttgcg
     3241 cttgcgtttt cccttgtcca gatagcccag tagctgacat tcatccgggg tcagcaccgt
     3301 ttctgcggac tggctttcta cgtgaaaagg atctaggtga agatcctttt tgataatctc
     3361 atgcctgaca tttatattcc ccagaacatc aggttaatgg cgtttttgat gtcattttcg
     3421 cggtggctga gatcagccac ttcttccccg ataacggaga ccggcacact ggccatatcg
     3481 gtggtcatca tgcgccagct ttcatccccg atatgcacca ccgggtaaag ttcacgggag
     3541 actttatctg acagcagacg tgcactggcc agggggatca ccatccgtcg ccccggcgtg
     3601 tcaataatat cactctgtac atccacaaac agacgataac ggctctctct tttataggtg
     3661 taaaccttaa actgccgtac gtataggctg cgcaactgtt gggaagggcg atcggtgcgg
     3721 gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg
     3781 gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga attgtaatac
     3841 gactcactat agggcgaatt gggccctcta gatgcatgct cgaggcagtg aaaaaaatgc
     3901 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa
     3961 caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag
     4021 gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg ctgattatga tcctctagag
     4081 tcggtgggcc tcgggggcgg gtgcggggtc ggcggggccg ccccgggtgg cttcggtcgg
     4141 agccatgggg tcgtgcgctc ctttcggtcg ggcgctgcgg gtcgtggggc gggcgtcagg
     4201 caccgggctt gcgggtcatg caccaggtgc gcggtccttc gggcacctcg acgtcggcgg
     4261 tgacggtgaa gccgagccgc tcgtagaagg ggaggttgcg gggcgcggag gtctccagga
     4321 aggcgggcac cccggcgcgc tcggccgcct ccactccggg gagcacgacg gcgctgccca
     4381 gacccttgcc ctggtggtcg ggcgagacgc cgacggtggc caggaaccac gcgggctcct
     4441 tgggccggtg cggcgccagg aggccttcca tctgttgctg cgcggccagc cgggaaccgc
     4501 tcaactcggc catgcgcggg ccgatctcgg cgaacaccgc ccccgcttcg acgctctccg
     4561 gcgtggtcca gaccgccacc gcggcgccgt cgtccgcgac ccacaccttg ccgatgtcga
     4621 gcccgacgcg cgtgaggaag agttcttgca gctcggtgac ccgctcgatg tggcggtccg
     4681 ggtcgacggt gtggcgcgtg gcggggtagt cggcgaacgc ggcggcgagg gtgcgtacgg
     4741 cccgggggac gtcgtcgcgg gtggcgaggc gcaccgtggg cttgtactcg gtcatggaag
     4801 gtcgtctcct tgtgaggggt caggggcgtg ggtcagggga tggtggcggc accggtcgtg
     4861 gcggccgacc tgcaggcatg caagctagct ttttgcaaaa gcctaggcct ccaaaaaagc
     4921 ctcctcacta cttctggaat agctcagagg ccgaggcggc ctcggcctct gcataaataa
     4981 aaaaaattag tcagccatgg ggcggagaat gggcggaact gggcggagtt aggggcggga
     5041 tgggcggagt taggggcggg actatggttg ctgactaatt gagatgcatg ctttgcatac
     5101 ttctgcctgc tggggagcct ggggactttc cacacctggt tgctgactaa ttgagatgca
     5161 tgctttgcat acttctgcct gctggggagc ctggggactt tccacaccct aactgacaca
     5221 ctcgagcggc cgccagtgtg atggatatct gcagaattcg ccctttgtac aaaaaagcag
     5281 gctttaaagg aaccaattca gtcgactgga tccgg
//
