LOCUS       sequence-71846-e        3150 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian U6-driven sgRNA (STm1) targeting GTCCCCTCCACCCCACAGTG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3150)
  AUTHORS   Esvelt KM, Mali P, Braff JL, Moosburner M, Yaung SJ, Church GM
  TITLE     Orthogonal Cas9 proteins for RNA-guided gene regulation and editing.
  JOURNAL   Nat Methods. 2013 Sep 29. doi: 10.1038/nmeth.2681.
  PUBMED    24076762
REFERENCE   2  (bases 1 to 3150)
  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. 2013 Sep 29. doi: 10.1038/nmeth.2681."
COMMENT     Sequence Label: M-ST1-sgRNA
FEATURES             Location/Qualifiers
     source          1..3150
                     /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        501..748
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     501..521
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     672..691
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        769..893
                     /label=St1 gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus 
                     thermophilus CRISPR/Cas9 system (Esvelt et al., 2013)"
     protein_bind    complement(905..929)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(974..990)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(974..990)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(987..1009)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    998..1014
                     /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(1022..1028,1029..1046,1047..1052))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1067..1088
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1205..1222)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1376..1964)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1456..1475)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2135..2950)
                     /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(2135..2950)
                     /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     2858..2877
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2951..3055)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3123..3141
                     /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 ttgtacaaaa aagcaggctt taaaggaacc aattcagtcg actggatccg
      481 gtaccaaggt cgggcaggaa gagggcctat ttcccatgat tccttcatat ttgcatatac
      541 gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca aagatattag
      601 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
      661 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
      721 tatatatctt gtggaaggac gaaacaccgt cccctccacc ccacagtggt ttttgtactc
      781 tcaagattta agtaactgta caacgaaact tacacagtta cttaaatctt gcagaagcta
      841 caaagataag gcttcatgcc gaaatcaaca ccctgtcatt ttatggcagg gtgttttttt
      901 ctagacccag ctttcttgta caaagttggc attagttcca tcccaatggc gcgccgagct
      961 tggctcgagc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     1021 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     1081 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     1141 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     1201 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     1261 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     1321 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     1381 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     1441 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     1501 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     1561 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     1621 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     1681 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     1741 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     1801 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     1861 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     1921 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     1981 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     2041 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     2101 tctaaagtat atatgagtaa acttggtctg acagttagaa aaactcatcg agcatcaaat
     2161 gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
     2221 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
     2281 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
     2341 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt
     2401 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
     2461 tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat
     2521 cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
     2581 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
     2641 tcccagggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
     2701 tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat
     2761 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
     2821 acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
     2881 ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa
     2941 tatggctcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     3001 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     3061 ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata
     3121 aaaataggcg tatcacgagg ccctttcgtc
//
