LOCUS       sequence-122865-        3455 bp    DNA     circular SYN 15-APR-2025
DEFINITION  gRNA targeting human HPRT gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3455)
  AUTHORS   Liao S, Tammaro M, Yan H
  TITLE     Enriching CRISPR-Cas9 targeted cells by co-targeting the HPRT gene.
  JOURNAL   Nucleic Acids Res. 2015 Jun 29. pii: gkv675.
  PUBMED    26130722
REFERENCE   2  (bases 1 to 3455)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res. 2015 Jun 29. pii: gkv675."
COMMENT     Sequence Label: HPRT2b-pGEM
FEATURES             Location/Qualifiers
     source          1..3455
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      33..42
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        121..369
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     121..141
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     292..311
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        390..465
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(477..501)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(580..598)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(581..598)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(616..632)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(616..632)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(629..651)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    640..656
                     /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(664..670,671..688,689..694))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    709..730
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(847..864)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1018..1606)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1098..1117)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1777..2568,2569..2637))
                     /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     2400..2419
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2638..2742)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2820..3275)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2957..2978)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3169..3188
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3401..3423
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3415..3432
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3416..3432
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(3439..3455,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(3439..3455,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggccgcggga ttgtacaaaa
       61 aagcaggctt taaaggaacc aattcagtcg actggatccg gtaccaaggt cgggcaggaa
      121 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
      181 ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      241 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      301 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      361 cgaaacaccg aagtaattca cttacagtcg ttttagagct agaaatagca agttaaaata
      421 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttctagaccc
      481 agctttcttg tacaaagttg gcattaatca ctagtgcggc cgcctgcagg tcgaccatat
      541 gggagagctc ccaacgcgtt ggatgcatag cttgagtatt ctatagtgtc acctaaatag
      601 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
      661 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
      721 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
      781 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
      841 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
      901 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
      961 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     1021 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     1081 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     1141 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     1201 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     1261 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     1321 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     1381 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     1441 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     1501 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     1561 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     1621 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     1681 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     1741 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     1801 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     1861 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     1921 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     1981 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     2041 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     2101 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     2161 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     2221 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     2281 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     2341 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     2401 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     2461 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     2521 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     2581 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     2641 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     2701 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     2761 gccacctgat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcagga
     2821 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     2881 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     2941 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     3001 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     3061 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     3121 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     3181 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     3241 acccgccgcg cttaatgcgc cgctacaggg cgcgtccatt cgccattcag gctgcgcaac
     3301 tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga
     3361 tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa
     3421 acgacggcca gtgaattgta atacgactca ctata
//
