LOCUS       sequence-96461-e        2799 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA expression by the H1 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2799)
  AUTHORS   Ranganathan V, Wahlin K, Maruotti J, Zack DJ
  TITLE     Expansion of the CRISPR-Cas9 genome targeting space through the use 
            of H1 promoter-expressed guide RNAs.
  JOURNAL   Nat Commun. 2014 Aug 8;5:4516. doi: 10.1038/ncomms5516.
  PUBMED    25105359
REFERENCE   2  (bases 1 to 2799)
  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: "2014-08-8"; volume: "5:4516. doi"; pages: " 
            10.1038/ncomms5516"
COMMENT     Sequence Label: pH1v1
FEATURES             Location/Qualifiers
     source          1..2799
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(17..33)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(17..33)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(30..52)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    41..57
                     /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(65..71,72..89,90..95))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    110..131
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(248..265)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(419..1007)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(499..518)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1178..1969,1970..2038))
                     /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     1801..1820
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2039..2143)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2211..2229
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2267..2289)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2389..2408
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2432..2646
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     2567..2586
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     misc_RNA        2658..2733
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(2745..2769)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
       61 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
      121 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
      181 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
      241 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
      301 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
      361 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
      421 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
      481 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
      541 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
      601 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
      661 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
      721 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
      781 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
      841 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
      901 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
      961 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     1021 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     1081 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     1141 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     1201 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     1261 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     1321 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     1381 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     1441 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     1501 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     1561 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     1621 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     1681 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     1741 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     1801 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     1861 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     1921 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     1981 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     2041 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     2101 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     2161 tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta
     2221 tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc
     2281 agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc
     2341 agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc
     2401 agattgtact gagagtgcac cataggaatt cgaacgctga cgtcatcaac ccgctccaag
     2461 gaatcgcggg cccagtgtca ctaggcggga acacccagcg cgcgtgcgcc ctggcaggaa
     2521 gatggctgtg agggacaggg gagtggcgcc ctgcaatatt tgcatgtcgc tatgtgttct
     2581 gggaaatcac cataaacgtg aaatgtcttt ggatttggga atcttataag ttctgtatga
     2641 gaccactttt tccctaggtt ttagagctag aaatagcaag ttaaaataag gctagtccgt
     2701 tatcaacttg aaaaagtggc accgagtcgg tgcttttttt ctagacccag ctttcttgta
     2761 caaagttggc attactagag tcgacctgca ggcatgcaa
//
