LOCUS       sequence-99467-e        2981 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR/Cas9 system gRNA cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2981)
  AUTHORS   Zheng Q, Cai X, Tan MH, Schaffert S, Arnold CP, Gong X, Chen CZ, 
            Huang S
  TITLE     Precise gene deletion and replacement using the CRISPR/Cas9 system 
            in human cells.
  JOURNAL   Biotechniques. 2014 Sep 1;57(3):115-24. doi: 10.2144/000114196. 
            eCollection 2014.
  PUBMED    25209046
REFERENCE   2  (bases 1 to 2981)
  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"; doi: "10.2144/000114196";
            journalName: "Biotechniques"; date: "2014-09-1- 1"; volume: "57"; 
            issue: "3"; pages: "115-24"
COMMENT     Sequence Label: pUC-H1-gRNA
FEATURES             Location/Qualifiers
     source          1..2981
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    106..127
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(142..147,148..165,166..172)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    180..196
                     /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     185..207
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     204..220
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     204..220
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_RNA        complement(253..328)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(424..443)
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     primer_bind     complement(585..602)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(585..601)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(594..616)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(810..829)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     929..951
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(989..1007)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1075..1179
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1180..1248,1249..2040)
                     /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(1398..1417)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2211..2799
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2700..2719
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2953..2970
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
       61 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
      121 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
      181 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttca
      241 cgcgtaaaaa aagcaccgac tcggtgccac tttttcaagt tgataacgga ctagccttat
      301 tttaacttgc tatttctagc tctaaaacag agaccactag tggcacaggt ctcaggggat
      361 ctgtggtcct catacagaac ttataagatt cccaaatcca aagacatttc acgtttatgg
      421 tgatttccca gaacacatag cgacatgcaa atattgcagg gcgccactcc cctgtccctc
      481 acagccatct tcctgccagg gcgcacgcgc gctgggtgtt cccgcctagt gacactgggc
      541 ccgcgattcc ttggagcggg ttgatgacgt cagcgttcga attcactggc cgtcgtttta
      601 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
      661 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
      721 cgcagcctga atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
      781 atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
      841 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
      901 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
      961 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     1021 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     1081 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     1141 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     1201 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     1261 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     1321 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     1381 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     1441 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     1501 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     1561 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     1621 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     1681 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     1741 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     1801 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     1861 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     1921 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     1981 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     2041 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     2101 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     2161 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     2221 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     2281 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     2341 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     2401 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     2461 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     2521 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     2581 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     2641 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     2701 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     2761 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     2821 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     2881 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     2941 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag a
//
