LOCUS       sequence-225153-        5315 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pPN10 with gRNA for spCRISPR-Cas9 against the 3' UTR of the DMPK 
            gene target: TGCGAACCAACGATAGGTG PAM: GGG.
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-gDM1d
FEATURES             Location/Qualifiers
     source          1..5315
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        51..299
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     51..71
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     222..241
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        320..395
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(407..431)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(516..534)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(517..534)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(552..568)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(552..568)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(565..587)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    576..592
                     /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(600..606,607..624,625..630))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    645..666
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(783..800)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(954..1542)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1034..1053)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1680..2054)
                     /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      2051..2060
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2260..3054)
                     /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(2260..3054)
                     /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(2371..2390)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2981..3000
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(3403..3705)
                     /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(3505..3524)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     3828..3850
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3842..3859
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3843..3859
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3866..3885
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3866..3884
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    3916..3997
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     3941..3960
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4227..4826)
                     /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(4310..4330)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     4807..4826
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(4923..5252)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4937..5072
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 taaaggaacc aattcagtcg actggatccg gtaccaaggt cgggcaggaa gagggcctat
       61 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattagaa
      121 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
      181 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg
      241 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg
      301 cacctatcgt tggttcgcag ttttagagct agaaatagca agttaaaata aggctagtcc
      361 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttctagaccc agctttcttg
      421 tacaaagttg gcattaaagg gcgaattcca gcacactggc ggccgttact agtggatccg
      481 agctcggtac caagcttgat gcatagcttg agtattctat agtgtcacct aaatagcttg
      541 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
      601 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
      661 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
      721 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
      781 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
      841 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
      901 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
      961 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     1021 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     1081 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     1141 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     1201 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     1261 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     1321 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     1381 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     1441 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     1501 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     1561 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     1621 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt tagcacgtgt
     1681 cagtcctgct cctcggccac gaagtgcacg cagttgccgg ccgggtcgcg cagggcgaac
     1741 tcccgccccc acggctgctc gccgatctcg gtcatggccg gcccggaggc gtcccggaag
     1801 ttcgtggaca cgacctccga ccactcggcg tacagctcgt ccaggccgcg cacccacacc
     1861 caggccaggg tgttgtccgg caccacctgg tcctggaccg cgctgatgaa cagggtcacg
     1921 tcgtcccgga ccacaccggc gaagtcgtcc tccacgaagt cccgggagaa cccgagccgg
     1981 tcggtccaga actcgaccgc tccggcgacg tcgcgcgcgg tgagcaccgg aacggcactg
     2041 gtcaacttgg ccatggtggc cctcctcacg tgctattatt gaagcattta tcagggttat
     2101 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     2161 cgcacatttc cccgaaaagt gccacctgat gcggtgtgaa ataccgcaca gatgcgtaag
     2221 gagaaaatac cgcatcagga aattgtaagc gttaataatt cagaagaact cgtcaagaag
     2281 gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg
     2341 gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg ctatgtcctg
     2401 atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc ggccattttc
     2461 caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct cgccgtcggg
     2521 catgctcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat gctcttcgtc
     2581 cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct cgatgcgatg
     2641 tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc
     2701 atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga gatcctgccc
     2761 cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt cgagcacagc
     2821 tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt cttgcagttc
     2881 attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct gcgctgacag
     2941 ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat agccgaatag
     3001 cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa tcatgcgaaa
     3061 cgatcctcat cctgtctctt gatcagagct tgatcccctg cgccatcaga tccttggcgg
     3121 caagaaagcc atccagttta ctttgcaggg cttcccaacc ttaccagagg gcgccccagc
     3181 tggcaattcc ggttcgcttg ctgtccataa aaccgcccag tctagctatc gccatgtaag
     3241 cccactgcaa gctacctgct ttctctttgc gcttgcgttt tcccttgtcc agatagccca
     3301 gtagctgaca ttcatccggg gtcagcaccg tttctgcgga ctggctttct acgtgaaaag
     3361 gatctaggtg aagatccttt ttgataatct catgcctgac atttatattc cccagaacat
     3421 caggttaatg gcgtttttga tgtcattttc gcggtggctg agatcagcca cttcttcccc
     3481 gataacggag accggcacac tggccatatc ggtggtcatc atgcgccagc tttcatcccc
     3541 gatatgcacc accgggtaaa gttcacggga gactttatct gacagcagac gtgcactggc
     3601 cagggggatc accatccgtc gccccggcgt gtcaataata tcactctgta catccacaaa
     3661 cagacgataa cggctctctc ttttataggt gtaaacctta aactgccgta cgtataggct
     3721 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
     3781 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
     3841 ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tgggccctct
     3901 agatgcatgc tcgaggcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc
     3961 tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt
     4021 tatgtttcag gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa
     4081 atgtggtatg gctgattatg atcctctaga gtcggtgggc ctcgggggcg ggtgcggggt
     4141 cggcggggcc gccccgggtg gcttcggtcg gagccatggg gtcgtgcgct cctttcggtc
     4201 gggcgctgcg ggtcgtgggg cgggcgtcag gcaccgggct tgcgggtcat gcaccaggtg
     4261 cgcggtcctt cgggcacctc gacgtcggcg gtgacggtga agccgagccg ctcgtagaag
     4321 gggaggttgc ggggcgcgga ggtctccagg aaggcgggca ccccggcgcg ctcggccgcc
     4381 tccactccgg ggagcacgac ggcgctgccc agacccttgc cctggtggtc gggcgagacg
     4441 ccgacggtgg ccaggaacca cgcgggctcc ttgggccggt gcggcgccag gaggccttcc
     4501 atctgttgct gcgcggccag ccgggaaccg ctcaactcgg ccatgcgcgg gccgatctcg
     4561 gcgaacaccg cccccgcttc gacgctctcc ggcgtggtcc agaccgccac cgcggcgccg
     4621 tcgtccgcga cccacacctt gccgatgtcg agcccgacgc gcgtgaggaa gagttcttgc
     4681 agctcggtga cccgctcgat gtggcggtcc gggtcgacgg tgtggcgcgt ggcggggtag
     4741 tcggcgaacg cggcggcgag ggtgcgtacg gcccggggga cgtcgtcgcg ggtggcgagg
     4801 cgcaccgtgg gcttgtactc ggtcatggaa ggtcgtctcc ttgtgagggg tcaggggcgt
     4861 gggtcagggg atggtggcgg caccggtcgt ggcggccgac ctgcaggcat gcaagctagc
     4921 tttttgcaaa agcctaggcc tccaaaaaag cctcctcact acttctggaa tagctcagag
     4981 gccgaggcgg cctcggcctc tgcataaata aaaaaaatta gtcagccatg gggcggagaa
     5041 tgggcggaac tgggcggagt taggggcggg atgggcggag ttaggggcgg gactatggtt
     5101 gctgactaat tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt
     5161 ccacacctgg ttgctgacta attgagatgc atgctttgca tacttctgcc tgctggggag
     5221 cctggggact ttccacaccc taactgacac actcgagcgg ccgccagtgt gatggatatc
     5281 tgcagaattc gccctttgta caaaaaagca ggctt
//
