LOCUS       sequence-225160-        5256 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5256)
  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 5256)
  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
FEATURES             Location/Qualifiers
     source          1..5256
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     50..70
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     221..240
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(347..371)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(456..474)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(457..474)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(492..508)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(492..508)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(505..527)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    516..532
                     /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(540..546,547..564,565..570))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    585..606
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(723..740)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(894..1482)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(974..993)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1620..1994)
                     /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      1991..2000
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2200..2994)
                     /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(2200..2994)
                     /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(2311..2330)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2921..2940
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(3343..3645)
                     /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(3445..3464)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     3768..3790
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3782..3799
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3783..3799
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3806..3825
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3806..3824
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    3856..3937
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     3881..3900
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4167..4766)
                     /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(4250..4270)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     4747..4766
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(4863..5192)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4877..5012
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 aaaggaacca attcagtcga ctggatccgg taccaaggtc gggcaggaag agggcctatt
       61 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattagaat
      121 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt
      181 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt
      241 aacttgaaag tatttcgatt tcttggcttt atatatctta agttaaaata aggctagtcc
      301 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttctagaccc agctttcttg
      361 tacaaagttg gcattaaagg gcgaattcca gcacactggc ggccgttact agtggatccg
      421 agctcggtac caagcttgat gcatagcttg agtattctat agtgtcacct aaatagcttg
      481 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
      541 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
      601 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
      661 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
      721 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
      781 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
      841 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
      901 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
      961 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     1021 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     1081 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     1141 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     1201 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     1261 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     1321 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     1381 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     1441 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     1501 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     1561 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt tagcacgtgt
     1621 cagtcctgct cctcggccac gaagtgcacg cagttgccgg ccgggtcgcg cagggcgaac
     1681 tcccgccccc acggctgctc gccgatctcg gtcatggccg gcccggaggc gtcccggaag
     1741 ttcgtggaca cgacctccga ccactcggcg tacagctcgt ccaggccgcg cacccacacc
     1801 caggccaggg tgttgtccgg caccacctgg tcctggaccg cgctgatgaa cagggtcacg
     1861 tcgtcccgga ccacaccggc gaagtcgtcc tccacgaagt cccgggagaa cccgagccgg
     1921 tcggtccaga actcgaccgc tccggcgacg tcgcgcgcgg tgagcaccgg aacggcactg
     1981 gtcaacttgg ccatggtggc cctcctcacg tgctattatt gaagcattta tcagggttat
     2041 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     2101 cgcacatttc cccgaaaagt gccacctgat gcggtgtgaa ataccgcaca gatgcgtaag
     2161 gagaaaatac cgcatcagga aattgtaagc gttaataatt cagaagaact cgtcaagaag
     2221 gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg
     2281 gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg ctatgtcctg
     2341 atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc ggccattttc
     2401 caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct cgccgtcggg
     2461 catgctcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat gctcttcgtc
     2521 cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct cgatgcgatg
     2581 tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc
     2641 atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga gatcctgccc
     2701 cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt cgagcacagc
     2761 tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt cttgcagttc
     2821 attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct gcgctgacag
     2881 ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat agccgaatag
     2941 cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa tcatgcgaaa
     3001 cgatcctcat cctgtctctt gatcagagct tgatcccctg cgccatcaga tccttggcgg
     3061 caagaaagcc atccagttta ctttgcaggg cttcccaacc ttaccagagg gcgccccagc
     3121 tggcaattcc ggttcgcttg ctgtccataa aaccgcccag tctagctatc gccatgtaag
     3181 cccactgcaa gctacctgct ttctctttgc gcttgcgttt tcccttgtcc agatagccca
     3241 gtagctgaca ttcatccggg gtcagcaccg tttctgcgga ctggctttct acgtgaaaag
     3301 gatctaggtg aagatccttt ttgataatct catgcctgac atttatattc cccagaacat
     3361 caggttaatg gcgtttttga tgtcattttc gcggtggctg agatcagcca cttcttcccc
     3421 gataacggag accggcacac tggccatatc ggtggtcatc atgcgccagc tttcatcccc
     3481 gatatgcacc accgggtaaa gttcacggga gactttatct gacagcagac gtgcactggc
     3541 cagggggatc accatccgtc gccccggcgt gtcaataata tcactctgta catccacaaa
     3601 cagacgataa cggctctctc ttttataggt gtaaacctta aactgccgta cgtataggct
     3661 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
     3721 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
     3781 ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tgggccctct
     3841 agatgcatgc tcgaggcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc
     3901 tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt
     3961 tatgtttcag gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa
     4021 atgtggtatg gctgattatg atcctctaga gtcggtgggc ctcgggggcg ggtgcggggt
     4081 cggcggggcc gccccgggtg gcttcggtcg gagccatggg gtcgtgcgct cctttcggtc
     4141 gggcgctgcg ggtcgtgggg cgggcgtcag gcaccgggct tgcgggtcat gcaccaggtg
     4201 cgcggtcctt cgggcacctc gacgtcggcg gtgacggtga agccgagccg ctcgtagaag
     4261 gggaggttgc ggggcgcgga ggtctccagg aaggcgggca ccccggcgcg ctcggccgcc
     4321 tccactccgg ggagcacgac ggcgctgccc agacccttgc cctggtggtc gggcgagacg
     4381 ccgacggtgg ccaggaacca cgcgggctcc ttgggccggt gcggcgccag gaggccttcc
     4441 atctgttgct gcgcggccag ccgggaaccg ctcaactcgg ccatgcgcgg gccgatctcg
     4501 gcgaacaccg cccccgcttc gacgctctcc ggcgtggtcc agaccgccac cgcggcgccg
     4561 tcgtccgcga cccacacctt gccgatgtcg agcccgacgc gcgtgaggaa gagttcttgc
     4621 agctcggtga cccgctcgat gtggcggtcc gggtcgacgg tgtggcgcgt ggcggggtag
     4681 tcggcgaacg cggcggcgag ggtgcgtacg gcccggggga cgtcgtcgcg ggtggcgagg
     4741 cgcaccgtgg gcttgtactc ggtcatggaa ggtcgtctcc ttgtgagggg tcaggggcgt
     4801 gggtcagggg atggtggcgg caccggtcgt ggcggccgac ctgcaggcat gcaagctagc
     4861 tttttgcaaa agcctaggcc tccaaaaaag cctcctcact acttctggaa tagctcagag
     4921 gccgaggcgg cctcggcctc tgcataaata aaaaaaatta gtcagccatg gggcggagaa
     4981 tgggcggaac tgggcggagt taggggcggg atgggcggag ttaggggcgg gactatggtt
     5041 gctgactaat tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt
     5101 ccacacctgg ttgctgacta attgagatgc atgctttgca tacttctgcc tgctggggag
     5161 cctggggact ttccacaccc taactgacac actcgagcgg ccgccagtgt gatggatatc
     5221 tgcagaattc gccctttgta caaaaaagca ggcttt
//
