LOCUS       sequence-275491-        8732 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A single vector mammalian expression system containing a CAG 
            promoter with Cas9 from Streptococcus thermophilus CRISPR1 (St1Cas9 
            LMD9:MTH17CL396 chimera) and its U6-driven sgRNA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8732)
  AUTHORS   Agudelo D, Carter S, Velimirovic M, Duringer A, Rivest JF, Levesque 
            S, Loehr J, Mouchiroud M, Cyr D, Waters PJ, Laplante M, Moineau S, 
            Goulet A, Doyon Y
  TITLE     Versatile and robust genome editing with Streptococcus thermophilus 
            CRISPR1-Cas9.
  JOURNAL   Genome Res. 2020 Jan;30(1):107-117. doi: 10.1101/gr.255414.119. Epub
            2020 Jan 3.
  PUBMED    31900288
REFERENCE   2  (bases 1 to 8732)
  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: 
            "10.1101/gr.255414.119"; journalName: "Genome Res"; date: "2020-01";
            volume: "30"; issue: "1"; pages: "107-117"
COMMENT     Sequence Label: U6_sgRNA_CAG_hSt1Cas9_MTH17CL396
FEATURES             Location/Qualifiers
     source          1..8732
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        421..724
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        726..998
                     /label=chicken beta-actin promoter
     intron          999..2015
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1939..1961
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2023..2042
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2108..2117
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2123..2143
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5537..5557
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(5661..5680)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5726..5781
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5780..5799)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(6142..6158)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6142..6158)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6155..6177)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6166..6182
                     /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(6190..6196,6197..6214,6215..6220))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6235..6256
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6315..6510
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6361..6496
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6516..6650
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6553..6572)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6607..6626
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6718..6735)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6889..7477)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6969..6988)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7648..8439,8440..8508))
                     /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     8271..8290
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8509..8613)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccc gagacgatta atgcgtctcg gtctttgtac tctggtacca gaagctacaa
      301 agataaggct tcatgccgaa atcaacaccc tgtcatttta tggcagggtg ttttttttac
      361 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
      421 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      481 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      541 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      601 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      661 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      721 catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc cctccccacc
      781 cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg
      841 ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg cggggcgagg cggagaggtg
      901 cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc
      961 ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc
     1021 gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt
     1081 tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat tagcgcttgg
     1141 tttaatgacg gcttgtttct tttctgtggc tgcgtgaaag ccttgagggg ctccgggagg
     1201 gccctttgtg cggggggagc ggctcggggg gtgcgtgcgt gtgtgtgtgc gtggggagcg
     1261 ccgcgtgcgg ctccgcgctg cccggcggct gtgagcgctg cgggcgcggc gcggggcttt
     1321 gtgcgctccg cagtgtgcgc gaggggagcg cggccggggg cggtgccccg cggtgcgggg
     1381 ggggctgcga ggggaacaaa ggctgcgtgc ggggtgtgtg cgtggggggg tgagcagggg
     1441 gtgtgggcgc gtcggtcggg ctgcaacccc ccctgcaccc ccctccccga gttgctgagc
     1501 acggcccggc ttcgggtgcg gggctccgta cggggcgtgg cgcggggctc gccgtgccgg
     1561 gcggggggtg gcggcaggtg ggggtgccgg gcggggcggg gccgcctcgg gccggggagg
     1621 gctcggggga ggggcgcggc ggcccccgga gcgccggcgg ctgtcgaggc gcggcgagcc
     1681 gcagccattg ccttttatgg taatcgtgcg agagggcgca gggacttcct ttgtcccaaa
     1741 tctgtgcgga gccgaaatct gggaggcgcc gccgcacccc ctctagcggg cgcggggcga
     1801 agcggtgcgg cgccggcagg aaggaaatgg gcggggaggg ccttcgtgcg tcgccgcgcc
     1861 gccgtcccct tctccctctc cagcctcggg gctgtccgcg gggggacggc tgccttcggg
     1921 ggggacgggg cagggcgggg ttcggcttct ggcgtgtgac cggcggctct agagcctctg
     1981 ctaaccatgt tcatgccttc ttctttttcc tacagctcct gggcaacgtg ctggttattg
     2041 tgctgtctca tcattttggc aaagaattct gcagtcgacg gtaccgcggg cccgggatcc
     2101 accggtcgcc accatgggcg ccccaaagaa gaagcggaag gtcggtatcc acggagtccc
     2161 agcagccagc gacctggtgc tgggcctgga catcggcatc ggcagcgtgg gcgtgggcat
     2221 cctgaacaag gtgaccggcg agatcatcca caagaacagt cgcatcttcc ctgctgctca
     2281 ggctgagaac aacctggtgc gccgcaccaa ccgccagggt cgccggcttg ctcgccgcaa
     2341 gaagcaccgg cgcgtgcgcc tgaaccgcct gttcgaggag agcggcctga tcaccgactt
     2401 caccaagatc agcatcaacc tgaaccccta ccagctgcgc gtgaagggcc tgaccgacga
     2461 gctgagcaac gaggagctgt tcatcgccct gaagaacatg gtgaagcacc gcggcatcag
     2521 ctacctggac gacgccagcg acgacggcaa cagcagcgtg ggcgactacg cccagatcgt
     2581 gaaggagaac agcaagcagc tggagaccaa gacccccggc cagatccagc tggagcgcta
     2641 ccagacctac ggccagctgc gcggcgactt caccgtggag aaggacggca agaagcaccg
     2701 cctgatcaac gtgttcccca ccagcgccta ccgcagcgag gccctgcgca tcctgcagac
     2761 ccagcaggag ttcaaccccc agatcaccga cgagttcatc aaccgctacc tggagatcct
     2821 gaccggcaag cgcaagtact accacggccc cggcaacgag aagagccgca ccgactacgg
     2881 ccgctaccgc accagcggcg agaccctgga caacatcttc ggcatcctga tcggcaagtg
     2941 caccttctac cccgacgagt tccgcgccgc caaggccagc tacaccgccc aggagttcaa
     3001 tctgctgaac gacctgaaca acctgaccgt gcccaccgag accaagaagc tgagcaagga
     3061 gcagaagaac cagatcatca actacgtgaa gaacgagaag gctatgggcc ccgccaagct
     3121 gttcaagtac atcgccaagc tgctgagctg cgacgtggcc gacatcaagg gctaccgcat
     3181 cgacaagagc ggcaaggccg agatccacac cttcgaggcc taccgcaaga tgaagaccct
     3241 ggagaccctg gacatcgagc agatggaccg agagaccctg gacaagctgg cctacgtgct
     3301 gaccctgaac accgagcgcg agggcatcca ggaggccctg gagcacgagt tcgccgacgg
     3361 cagcttcagc cagaaacagg tggacgagct ggtgcagttc cgcaaggcca acagcagcat
     3421 cttcggcaag ggctggcaca acttcagcgt gaagctgatg atggagctga tccccgagct
     3481 gtacgagacc agcgaggagc agatgaccat cctgacccgc ctgggcaagc agaagaccac
     3541 cagcagcagc aacaagacca agtacatcga cgagaagctg ctgaccgagg agatctacaa
     3601 ccccgtggtg gccaagagcg tgcgccaggc catcaagatc gtgaacgccg ccatcaagga
     3661 gtacggcgac ttcgacaaca tcgtgatcga gatggcccgc gagaccaacg aggacgacga
     3721 gaagaaggcc atccagaaga tccagaaggc caacaaggac gagaaggacg ccgccatgct
     3781 gaaggccgcc aaccagtaca acggcaaggc cgagctgccc cacagcgtgt tccacggcca
     3841 caagcagctg gccaccaaga tccgcctgtg gcaccagcag ggcgagcgct gcctgtacac
     3901 cggcaagacc atcagcatcc acgacctgat caacaacagc aaccagttcg aggtggacca
     3961 catcctgccc ctgagcatca ccttcgacga cagcctggcc aacaaggtgc tggtgtacgc
     4021 caccgccaac caggagaagg gccagcgcac cccctaccag gccctggaca gcatggacga
     4081 cgcctggagc ttccgcgagc tgaaggcctt cgtgcgcgag agcaagaccc tgagcaacaa
     4141 gaagaaggag tatctgctga ccgaggagga catcagcaag ttcgacgtgc gcaagaagtt
     4201 catcgagcgc aacctggtgg acacccgcta cgccagccgc gtggtgctga acgccctgca
     4261 ggagcacttc cgcgcccaca agatcgacac caaggtgagc gtggtgcgcg gccagttcac
     4321 cagccagctg cgccgccact ggggcatcga gaagacccgc gacacctacc accaccacgc
     4381 cgtggacgcc ctgatcattg cggcttctag ccagctgaac ctgtggaaga agcagaagaa
     4441 caccctggtg agctacagcg aggaccagct gctggacatc gagaccggcg agctgatcag
     4501 cgacgacgag tacaaggaga gcgtgttcaa ggccccctac cagcacttcg tggacaccct
     4561 gaagagcaag gagttcgagg acagcatcct gttcagctac caggtggaca gcaagttcaa
     4621 ccgcaagatc agcgacgcca ccatctacgc cacccgccag gccaaggtgg gcaaggacaa
     4681 ggccgacgag acctacgtgc tgggcaagat caaggacatc tacacccagg acggctacga
     4741 cgccttcatg aagatctaca agaaggacaa gagcaagttc ctgatgtacc gccacgaccc
     4801 ccagaccttc gagaaggtga tcgagcccat cctggagaac taccccaaca agcagatcaa
     4861 cgagaaaggc aaggaggtgc cctgcaaccc cttcctgaag tacaaggagg agcacggcta
     4921 catccgcaag tacagcaaga agggcaacgg ccccgagatc aagagcctga agtactacga
     4981 cagcaagctg ggcaaccaca tcgacatcac ccccaaggac agcaacaaca aggtggtgct
     5041 gcagagcctt aagccctggc gcaccgacgt gtacttcaac aagaacaccg gcaagtacga
     5101 gatcctgggg ctgaagtaca gcgatatgca gtttgagaaa ggcacaggca agtacagcat
     5161 cagcaaggag cagtacgaga acatcaaggt gcgcgagggc gtggacgaga acagcgagtt
     5221 caagttcacc ctgtacaaga acgaccttct gctgctgaag gacagcgaga acggcgagca
     5281 aatcctgctg cgcttcacca gccgcaacga caccagcaag cactacgtgg agctgaagcc
     5341 ctacaaccgc cagaagttcg agggcagcga gtacctgatc aagagcctgg gcaccgtggc
     5401 caagggcggc cagtgcatca agggcctggg caagagcaac atcagcatct acaaggtgcg
     5461 caccgacgtg ctgggcaacc agcacatcat caagaacgag ggcgacaagc ccaagttgga
     5521 cttcagcagg gctgacccca agaagaagag gaaggtggga tcctgataag aattcgcggc
     5581 cgcactcctc aggtgcaggc tgcctatcag aaggtggtgg ctggtgtggc caatgccctg
     5641 gctcacaaat accactgaga tctttttccc tctgccaaaa attatgggga catcatgaag
     5701 ccccttgagc atctgacttc tggctaataa aggaaattta ttttcattgc aatagtgtgt
     5761 tggaattttt tgtgtctctc actcggaagg acatatggga gggcaaatca tttaaaacat
     5821 cagaatgagt atttggttta gagtttggca acatatgccc atatgctggc tgccatgaac
     5881 aaaggttggc tataaagagg tcatcagtat atgaaacagc cccctgctgt ccattcctta
     5941 ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt tgtgttattt
     6001 ttttctttaa catccctaaa attttcctta catgttttac tagccagatt tttcctcctc
     6061 tcctgactac tcccagtcat agctgtccct cttctcttat ggagatccct cgacctgcag
     6121 cccaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     6181 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     6241 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     6301 gtgccagcgg atccgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     6361 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     6421 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     6481 ggcttttttg gaggcctagg cttttgcaaa aagctaactt gtttattgca gcttataatg
     6541 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     6601 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc cgctgcatta
     6661 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     6721 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     6781 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     6841 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     6901 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     6961 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     7021 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     7081 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     7141 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     7201 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     7261 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     7321 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     7381 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     7441 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     7501 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     7561 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     7621 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     7681 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     7741 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     7801 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     7861 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     7921 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     7981 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     8041 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     8101 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     8161 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     8221 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     8281 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     8341 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     8401 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     8461 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     8521 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     8581 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctgg
     8641 gtcgacattg attattgact agctgtacaa aaaagcaggc tttaaaggaa ccaattcagt
     8701 cgactggatc cggtaccaag gtcgggcagg aa
//
