LOCUS       sequence-193521-        3046 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CCR5-targeting gRNA expression plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3046)
  AUTHORS   Chen X, Janssen JM, Liu J, Maggio I, 't Jong AEJ, Mikkers HMM, 
            Goncalves MAFV
  TITLE     In trans paired nicking triggers seamless genome editing without 
            double-stranded DNA cutting.
  JOURNAL   Nat Commun. 2017 Sep 22;8(1):657. doi: 10.1038/s41467-017-00687-1.
  PUBMED    28939824
REFERENCE   2  (bases 1 to 3046)
  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: "2017-09-22"; volume: "8(1):657. doi"; pages: " 
            10.1038/s41467-017-00687-1"
COMMENT     Sequence Label: AY23_pgRNA.R5.2
FEATURES             Location/Qualifiers
     source          1..3046
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..22
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(37..42,43..60,61..67)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    75..91
                     /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     80..102
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     99..115
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     99..115
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        187..435
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     187..207
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     358..377
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        456..531
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(545..562)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(545..561)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(554..576)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(770..789)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     889..911
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(949..967)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1035..1139
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1140..1208,1209..2000)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1358..1377)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2171..2759
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2660..2679
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2913..2930
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
       61 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
      121 ttacgccaag cttccatggg atatcgcatg cctgcagagc tctagagtcc ggccgccccc
      181 ttcaccgagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt
      241 agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga
      301 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac
      361 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg
      421 aaaggacgaa acaccgagcc aagctctcca tctaggtttt agagctagaa atagcaagtt
      481 aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttttttga
      541 attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
      601 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
      661 atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgcctgatg cggtattttc
      721 tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct
      781 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
      841 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
      901 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
      961 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     1021 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     1081 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     1141 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     1201 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     1261 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     1321 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     1381 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     1441 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     1501 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     1561 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     1621 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     1681 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     1741 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     1801 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     1861 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     1921 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     1981 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     2041 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     2101 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     2161 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     2221 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     2281 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     2341 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     2401 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     2461 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     2521 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     2581 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     2641 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     2701 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     2761 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     2821 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     2881 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     2941 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     3001 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaat
//
