LOCUS       sequence-263807-        8727 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 8727)
  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 8727)
  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..8727
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        93..341
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     93..113
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     264..283
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        513..816
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        819..1094
                     /label=chicken beta-actin promoter
     intron          1095..2103
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2027..2049
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2111..2130
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2196..2205
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2211..2231
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5625..5645
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(5749..5768)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5814..5869
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5868..5887)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(6230..6246)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6230..6246)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6243..6265)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6254..6270
                     /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(6278..6284,6285..6302,6303..6308))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6323..6344
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6403..6598
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6449..6584
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6604..6738
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6641..6660)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6695..6714
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6806..6823)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6977..7565)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7057..7076)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7736..8527,8528..8596))
                     /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     8359..8378
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8597..8701)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agctgtacaa aaaagcaggc tttaaaggaa ccaattcagt
       61 cgactggatc cggtaccaag gtcgggcagg aagagggcct atttcccatg attccttcat
      121 atttgcatat acgatacaag gctgttagag agataattag aattaatttg actgtaaaca
      181 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
      241 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
      301 atttcttggc tttatatatc ttgtggaaag gacgaaacac ccgagacgat taatgcgtct
      361 cggtctttgt actctggtac cagaagctac aaagataagg cttcatgccg aaatcaacac
      421 cctgtcattt tatggcaggg tgtttttttt actagttatt aatagtaatc aattacgggg
      481 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
      541 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
      601 gtaacgccaa tagggacttt ccattgacgt caatgggtgg actatttacg gtaaactgcc
      661 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
      721 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
      781 cagtacatct acgtattagt catcgctatt accatgggtc gaggtgagcc ccacgttctg
      841 cttcactctc cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta
      901 attattttgt gcagcgatgg gggcgggggg ggggggggcg cgcgccaggc ggggcggggc
      961 ggggcgaggg gcggggcggg gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg
     1021 cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa
     1081 gcgcgcggcg ggcgggagtc gctgcgttgc cttcgccccg tgccccgctc cgcgccgcct
     1141 cgcgccgccc gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg
     1201 gcccttctcc tccgggctgt aattagcgct tggtttaatg acggctcgtt tcttttctgt
     1261 ggctgcgtga aagccttaaa gggctccggg agggcccttt gtgcgggggg gagcggctcg
     1321 gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggcccgcg ctgcccggcg
     1381 gctgtgagcg ctgcgggcgc ggcgcggggc tttgtgcgct ccgcgtgtgc gcgaggggag
     1441 cgcggccggg ggcggtgccc cgcggtgcgg gggggctgcg aggggaacaa aggctgcgtg
     1501 cggggtgtgt gcgtgggggg gtgagcaggg ggtgtgggcg cggcggtcgg gctgtaaccc
     1561 ccccctgcac ccccctcccc gagttgctga gcacggcccg gcttcgggtg cggggctccg
     1621 tgcggggcgt ggcgcggggc tcgccgtgcc gggcgggggg tggcggcagg tgggggtgcc
     1681 gggcggggcg gggccgcctc gggccgggga gggctcgggg gaggggcgcg gcggccccgg
     1741 agcgccggcg gctgtcgagg cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc
     1801 gagagggcgc agggacttcc tttgtcccaa atctggcgga gccgaaatct gggaggcgcc
     1861 gccgcacccc ctctagcggg cgcgggcgaa gcggtgcggc gccggcagga aggaaatggg
     1921 cggggagggc cttcgtgcgt cgccgcgccg ccgtcccctt ctccatctcc agcctcgggg
     1981 ctgccgcagg gggacggctg ccttcggggg ggacggggca gggcggggtt cggcttctgg
     2041 cgtgtgaccg gcggctctag agcctctgct aaccatgttc atgccttctt ctttttccta
     2101 cagctcctgg gcaacgtgct ggttattgtg ctgtctcatc attttggcaa agaattctgc
     2161 agtcgacggt accgcgggcc cgggatccac cggtcgccac catgggcgcc ccaaagaaga
     2221 agcggaaggt cggtatccac ggagtcccag cagccagcga cctggtgctg ggcctggaca
     2281 tcggcatcgg cagcgtgggc gtgggcatcc tgaacaaggt gaccggcgag atcatccaca
     2341 agaacagtcg catcttccct gctgctcagg ctgagaacaa cctggtgcgc cgcaccaacc
     2401 gccagggtcg ccggcttgct cgccgcaaga agcaccggcg cgtgcgcctg aaccgcctgt
     2461 tcgaggagag cggcctgatc accgacttca ccaagatcag catcaacctg aacccctacc
     2521 agctgcgcgt gaagggcctg accgacgagc tgagcaacga ggagctgttc atcgccctga
     2581 agaacatggt gaagcaccgc ggcatcagct acctggacga cgccagcgac gacggcaaca
     2641 gcagcgtggg cgactacgcc cagatcgtga aggagaacag caagcagctg gagaccaaga
     2701 cccccggcca gatccagctg gagcgctacc agacctacgg ccagctgcgc ggcgacttca
     2761 ccgtggagaa ggacggcaag aagcaccgcc tgatcaacgt gttccccacc agcgcctacc
     2821 gcagcgaggc cctgcgcatc ctgcagaccc agcaggagtt caacccccag atcaccgacg
     2881 agttcatcaa ccgctacctg gagatcctga ccggcaagcg caagtactac cacggccccg
     2941 gcaacgagaa gagccgcacc gactacggcc gctaccgcac cagcggcgag accctggaca
     3001 acatcttcgg catcctgatc ggcaagtgca ccttctaccc cgacgagttc cgcgccgcca
     3061 aggccagcta caccgcccag gagttcaatc tgctgaacga cctgaacaac ctgaccgtgc
     3121 ccaccgagac caagaagctg agcaaggagc agaagaacca gatcatcaac tacgtgaaga
     3181 acgagaaggc tatgggcccc gccaagctgt tcaagtacat cgccaagctg ctgagctgcg
     3241 acgtggccga catcaagggc taccgcatcg acaagagcgg caaggccgag atccacacct
     3301 tcgaggccta ccgcaagatg aagaccctgg agaccctgga catcgagcag atggaccgag
     3361 agaccctgga caagctggcc tacgtgctga ccctgaacac cgagcgcgag ggcatccagg
     3421 aggccctgga gcacgagttc gccgacggca gcttcagcca gaaacaggtg gacgagctgg
     3481 tgcagttccg caaggccaac agcagcatct tcggcaaggg ctggcacaac ttcagcgtga
     3541 agctgatgat ggagctgatc cccgagctgt acgagaccag cgaggagcag atgaccatcc
     3601 tgacccgcct gggcaagcag aagaccacca gcagcagcaa caagaccaag tacatcgacg
     3661 agaagctgct gaccgaggag atctacaacc ccgtggtggc caagagcgtg cgccaggcca
     3721 tcaagatcgt gaacgccgcc atcaaggagt acggcgactt cgacaacatc gtgatcgaga
     3781 tggcccgcga gaccaacgag gacgacgaga agaaggccat ccagaagatc cagaaggcca
     3841 acaaggacga gaaggacgcc gccatgctga aggccgccaa ccagtacaac ggcaaggccg
     3901 agctgcccca cagcgtgttc cacggccaca agcagctggc caccaagatc cgcctgtggc
     3961 accagcaggg cgagcgctgc ctgtacaccg gcaagaccat cagcatccac gacctgatca
     4021 acaacagcaa ccagttcgag gtggaccaca tcctgcccct gagcatcacc ttcgacgaca
     4081 gcctggccaa caaggtgctg gtgtacgcca ccgccaacca ggagaagggc cagcgcaccc
     4141 cctaccaggc cctggacagc atggacgacg cctggagctt ccgcgagctg aaggccttcg
     4201 tgcgcgagag caagaccctg agcaacaaga agaaggagta tctgctgacc gaggaggaca
     4261 tcagcaagtt cgacgtgcgc aagaagttca tcgagcgcaa cctggtggac acccgctacg
     4321 ccagccgcgt ggtgctgaac gccctgcagg agcacttccg cgcccacaag atcgacacca
     4381 aggtgagcgt ggtgcgcggc cagttcacca gccagctgcg ccgccactgg ggcatcgaga
     4441 agacccgcga cacctaccac caccacgccg tggacgccct gatcattgcg gcttctagcc
     4501 agctgaacct gtggaagaag cagaagaaca ccctggtgag ctacagcgag gaccagctgc
     4561 tggacatcga gaccggcgag ctgatcagcg acgacgagta caaggagagc gtgttcaagg
     4621 ccccctacca gcacttcgtg gacaccctga agagcaagga gttcgaggac agcatcctgt
     4681 tcagctacca ggtggacagc aagttcaacc gcaagatcag cgacgccacc atctacgcca
     4741 cccgccaggc caaggtgggc aaggacaagg ccgacgagac ctacgtgctg ggcaagatca
     4801 aggacatcta cacccaggac ggctacgacg ccttcatgaa gatctacaag aaggacaaga
     4861 gcaagttcct gatgtaccgc cacgaccccc agaccttcga gaaggtgatc gagcccatcc
     4921 tggagaacta ccccaacaag cagatcaacg agaaaggcaa ggaggtgccc tgcaacccct
     4981 tcctgaagta caaggaggag cacggctaca tccgcaagta cagcaagaag ggcaacggcc
     5041 ccgagatcaa gagcctgaag tactacgaca gcaagctggg caaccacatc gacatcaccc
     5101 ccaaggacag caacaacaag gtggtgctgc agagccttaa gccctggcgc accgacgtgt
     5161 acttcaacaa gaacaccggc aagtacgaga tcctggggct gaagtacagc gatatgcagt
     5221 ttgagaaagg cacaggcaag tacagcatca gcaaggagca gtacgagaac atcaaggtgc
     5281 gcgagggcgt ggacgagaac agcgagttca agttcaccct gtacaagaac gaccttctgc
     5341 tgctgaagga cagcgagaac ggcgagcaaa tcctgctgcg cttcaccagc cgcaacgaca
     5401 ccagcaagca ctacgtggag ctgaagccct acaaccgcca gaagttcgag ggcagcgagt
     5461 acctgatcaa gagcctgggc accgtggcca agggcggcca gtgcatcaag ggcctgggca
     5521 agagcaacat cagcatctac aaggtgcgca ccgacgtgct gggcaaccag cacatcatca
     5581 agaacgaggg cgacaagccc aagttggact tcagcagggc tgaccccaag aagaagagga
     5641 aggtgggatc ctgataagaa ttcgcggccg cactcctcag gtgcaggctg cctatcagaa
     5701 ggtggtggct ggtgtggcca atgccctggc tcacaaatac cactgagatc tttttccctc
     5761 tgccaaaaat tatggggaca tcatgaagcc ccttgagcat ctgacttctg gctaataaag
     5821 gaaatttatt ttcattgcaa tagtgtgttg gaattttttg tgtctctcac tcggaaggac
     5881 atatgggagg gcaaatcatt taaaacatca gaatgagtat ttggtttaga gtttggcaac
     5941 atatgcccat atgctggctg ccatgaacaa aggttggcta taaagaggtc atcagtatat
     6001 gaaacagccc cctgctgtcc attccttatt ccatagaaaa gccttgactt gaggttagat
     6061 tttttttata ttttgttttg tgttattttt ttctttaaca tccctaaaat tttccttaca
     6121 tgttttacta gccagatttt tcctcctctc ctgactactc ccagtcatag ctgtccctct
     6181 tctcttatga agatccctcg acctgcagcc caagcttggc gtaatcatgg tcatagctgt
     6241 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     6301 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     6361 tgcccgcttt ccagtcggga aacctgtcgt gccagcggat ccgcatctca attagtcagc
     6421 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     6481 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
     6541 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     6601 gctaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     6661 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     6721 tcttatcatg tctggatccg ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     6781 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     6841 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     6901 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     6961 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     7021 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     7081 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     7141 ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg
     7201 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     7261 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     7321 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     7381 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     7441 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     7501 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     7561 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     7621 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     7681 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     7741 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     7801 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     7861 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     7921 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     7981 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     8041 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     8101 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     8161 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     8221 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     8281 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     8341 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     8401 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     8461 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     8521 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     8581 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     8641 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     8701 gcacatttcc ccgaaaagtg ccacctg
//
