LOCUS       sequence-225214-        5315 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pPN10 with gRNA for spCRISPR-Cas9 targeted to CAG repeat,  PAM: CAG.
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-gCAG
FEATURES             Location/Qualifiers
     source          1..5315
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        46..294
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     46..66
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     217..236
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        315..390
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(402..426)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(511..529)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(512..529)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(547..563)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(547..563)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(560..582)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    571..587
                     /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(595..601,602..619,620..625))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    640..661
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(778..795)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(949..1537)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1029..1048)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1675..2049)
                     /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      2046..2055
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2255..3049)
                     /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(2255..3049)
                     /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(2366..2385)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2976..2995
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(3398..3700)
                     /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(3500..3519)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     3823..3845
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3837..3854
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3838..3854
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3861..3880
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3861..3879
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    3911..3992
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     3936..3955
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4222..4821)
                     /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(4305..4325)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     4802..4821
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(4918..5247)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4932..5067
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 gaaccaattc agtcgactgg atccggtacc aaggtcgggc aggaagaggg cctatttccc
       61 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tagaattaat
      121 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
      181 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
      241 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggcagc
      301 agcagcagca gcaggtttta gagctagaaa tagcaagtta aaataaggct agtccgttat
      361 caacttgaaa aagtggcacc gagtcggtgc tttttttcta gacccagctt tcttgtacaa
      421 agttggcatt aaagggcgaa ttccagcaca ctggcggccg ttactagtgg atccgagctc
      481 ggtaccaagc ttgatgcata gcttgagtat tctatagtgt cacctaaata gcttggcgta
      541 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
      601 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
      661 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
      721 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
      781 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
      841 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
      901 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
      961 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     1021 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     1081 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     1141 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     1201 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     1261 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     1321 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     1381 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     1441 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     1501 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     1561 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     1621 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttagca cgtgtcagtc
     1681 ctgctcctcg gccacgaagt gcacgcagtt gccggccggg tcgcgcaggg cgaactcccg
     1741 cccccacggc tgctcgccga tctcggtcat ggccggcccg gaggcgtccc ggaagttcgt
     1801 ggacacgacc tccgaccact cggcgtacag ctcgtccagg ccgcgcaccc acacccaggc
     1861 cagggtgttg tccggcacca cctggtcctg gaccgcgctg atgaacaggg tcacgtcgtc
     1921 ccggaccaca ccggcgaagt cgtcctccac gaagtcccgg gagaacccga gccggtcggt
     1981 ccagaactcg accgctccgg cgacgtcgcg cgcggtgagc accggaacgg cactggtcaa
     2041 cttggccatg gtggccctcc tcacgtgcta ttattgaagc atttatcagg gttattgtct
     2101 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     2161 atttccccga aaagtgccac ctgatgcggt gtgaaatacc gcacagatgc gtaaggagaa
     2221 aataccgcat caggaaattg taagcgttaa taattcagaa gaactcgtca agaaggcgat
     2281 agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg aagcggtcag
     2341 cccattcgcc gccaagctct tcagcaatat cacgggtagc caacgctatg tcctgatagc
     2401 ggtccgccac acccagccgg ccacagtcga tgaatccaga aaagcggcca ttttccacca
     2461 tgatattcgg caagcaggca tcgccatggg tcacgacgag atcctcgccg tcgggcatgc
     2521 tcgccttgag cctggcgaac agttcggctg gcgcgagccc ctgatgctct tcgtccagat
     2581 catcctgatc gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg
     2641 cttggtggtc gaatgggcag gtagccggat caagcgtatg cagccgccgc attgcatcag
     2701 ccatgatgga tactttctcg gcaggagcaa ggtgagatga caggagatcc tgccccggca
     2761 cttcgcccaa tagcagccag tcccttcccg cttcagtgac aacgtcgagc acagctgcgc
     2821 aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc ctcgtcttgc agttcattca
     2881 gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga
     2941 acacggcggc atcagagcag ccgattgtct gttgtgccca gtcatagccg aatagcctct
     3001 ccacccaagc ggccggagaa cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc
     3061 ctcatcctgt ctcttgatca gagcttgatc ccctgcgcca tcagatcctt ggcggcaaga
     3121 aagccatcca gtttactttg cagggcttcc caaccttacc agagggcgcc ccagctggca
     3181 attccggttc gcttgctgtc cataaaaccg cccagtctag ctatcgccat gtaagcccac
     3241 tgcaagctac ctgctttctc tttgcgcttg cgttttccct tgtccagata gcccagtagc
     3301 tgacattcat ccggggtcag caccgtttct gcggactggc tttctacgtg aaaaggatct
     3361 aggtgaagat cctttttgat aatctcatgc ctgacattta tattccccag aacatcaggt
     3421 taatggcgtt tttgatgtca ttttcgcggt ggctgagatc agccacttct tccccgataa
     3481 cggagaccgg cacactggcc atatcggtgg tcatcatgcg ccagctttca tccccgatat
     3541 gcaccaccgg gtaaagttca cgggagactt tatctgacag cagacgtgca ctggccaggg
     3601 ggatcaccat ccgtcgcccc ggcgtgtcaa taatatcact ctgtacatcc acaaacagac
     3661 gataacggct ctctctttta taggtgtaaa ccttaaactg ccgtacgtat aggctgcgca
     3721 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
     3781 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta
     3841 aaacgacggc cagtgaattg taatacgact cactataggg cgaattgggc cctctagatg
     3901 catgctcgag gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
     3961 ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt
     4021 ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg
     4081 gtatggctga ttatgatcct ctagagtcgg tgggcctcgg gggcgggtgc ggggtcggcg
     4141 gggccgcccc gggtggcttc ggtcggagcc atggggtcgt gcgctccttt cggtcgggcg
     4201 ctgcgggtcg tggggcgggc gtcaggcacc gggcttgcgg gtcatgcacc aggtgcgcgg
     4261 tccttcgggc acctcgacgt cggcggtgac ggtgaagccg agccgctcgt agaaggggag
     4321 gttgcggggc gcggaggtct ccaggaaggc gggcaccccg gcgcgctcgg ccgcctccac
     4381 tccggggagc acgacggcgc tgcccagacc cttgccctgg tggtcgggcg agacgccgac
     4441 ggtggccagg aaccacgcgg gctccttggg ccggtgcggc gccaggaggc cttccatctg
     4501 ttgctgcgcg gccagccggg aaccgctcaa ctcggccatg cgcgggccga tctcggcgaa
     4561 caccgccccc gcttcgacgc tctccggcgt ggtccagacc gccaccgcgg cgccgtcgtc
     4621 cgcgacccac accttgccga tgtcgagccc gacgcgcgtg aggaagagtt cttgcagctc
     4681 ggtgacccgc tcgatgtggc ggtccgggtc gacggtgtgg cgcgtggcgg ggtagtcggc
     4741 gaacgcggcg gcgagggtgc gtacggcccg ggggacgtcg tcgcgggtgg cgaggcgcac
     4801 cgtgggcttg tactcggtca tggaaggtcg tctccttgtg aggggtcagg ggcgtgggtc
     4861 aggggatggt ggcggcaccg gtcgtggcgg ccgacctgca ggcatgcaag ctagcttttt
     4921 gcaaaagcct aggcctccaa aaaagcctcc tcactacttc tggaatagct cagaggccga
     4981 ggcggcctcg gcctctgcat aaataaaaaa aattagtcag ccatggggcg gagaatgggc
     5041 ggaactgggc ggagttaggg gcgggatggg cggagttagg ggcgggacta tggttgctga
     5101 ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca
     5161 cctggttgct gactaattga gatgcatgct ttgcatactt ctgcctgctg gggagcctgg
     5221 ggactttcca caccctaact gacacactcg agcggccgcc agtgtgatgg atatctgcag
     5281 aattcgccct ttgtacaaaa aagcaggctt taaag
//
