LOCUS       sequence-77412-e        2792 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For in vitro transcription of sgRNA from the T7 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2792)
  AUTHORS   Shen B, Zhang W, Zhang J, Zhou J, Wang J, Chen L, Wang L, Hodgkins 
            A, Iyer V, Huang X, Skarnes WC
  TITLE     Efficient genome modification by CRISPR-Cas9 nickase with minimal 
            off-target effects.
  JOURNAL   Nat Methods. 2014 Mar 2. doi: 10.1038/nmeth.2857.
  PUBMED    24584192
REFERENCE   2  (bases 1 to 2792)
  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 
            Methods. 2014 Mar 2. doi: 10.1038/nmeth.2857."
COMMENT     Sequence Label: pUC57-sgRNA expression vector
FEATURES             Location/Qualifiers
     source          1..2792
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        442..460
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        482..557
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(615..631)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(615..631)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(628..650)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    639..655
                     /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(663..669,670..687,688..693))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    708..729
                     /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(1777..2592)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1777..2592)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2500..2519
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2593..2697)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2765..2783
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa
      421 tgcatctaga tatcggatcc ctaatacgac tcactatagg tgagaccgag agagggtctc
      481 agttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag
      541 tggcaccgag tcggtgcttt ttttaaaggg cccgtcgact gcagaggcct gcatgcaagc
      601 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
      661 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
      721 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
      781 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgcggccgc
      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 tgacagttag aaaaactcat cgagcatcaa
     1801 atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt
     1861 ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg
     1921 gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat
     1981 aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag
     2041 tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc
     2101 actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg
     2161 atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc
     2221 cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt
     2281 tttcccaggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt
     2341 gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac
     2401 atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc
     2461 atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc
     2521 atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg
     2581 aatatggctc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     2641 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     2701 atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta
     2761 taaaaatagg cgtatcacga ggccctttcg tc
//
