LOCUS       sequence-273341-        8735 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:TH1477 chimera) and its U6-driven sgRNA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8735)
  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 8735)
  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_TH1477
FEATURES             Location/Qualifiers
     source          1..8735
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        94..342
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     94..114
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     265..284
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        514..817
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          1095..2111
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2035..2057
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2119..2138
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2204..2213
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2219..2239
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5633..5653
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(5757..5776)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5822..5877
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5876..5895)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(6238..6254)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6238..6254)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6251..6273)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6262..6278
                     /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(6286..6292,6293..6310,6311..6316))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6331..6352
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6411..6606
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6457..6592
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6612..6746
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6649..6668)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6703..6722
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6814..6831)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6985..7573)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7065..7084)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7744..8535,8536..8604))
                     /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     8367..8386
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8605..8709)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagctgtaca aaaaagcagg ctttaaagga accaattcag
       61 tcgactggat ccggtaccaa ggtcgggcag gaagagggcc tatttcccat gattccttca
      121 tatttgcata tacgatacaa ggctgttaga gagataatta gaattaattt gactgtaaac
      181 acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca
      241 gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc
      301 gatttcttgg ctttatatat cttgtggaaa ggacgaaaca cccgagacga ttaatgcgtc
      361 tcggtctttg tactctggta ccagaagcta caaagataag gcttcatgcc gaaatcaaca
      421 ccctgtcatt ttatggcagg gtgttttttt tactagttat taatagtaat caattacggg
      481 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      541 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      601 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      661 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
      721 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
      781 gcagtacatc tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg
      841 cttcactctc cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta
      901 attattttgt gcagcgatgg gggcgggggg ggggggggsg cgcgccrggs gggggggggs
      961 ggggsgrggg gsggggsggg gsgaggcgga gaggtgcggc ggcagccaat cagagcggcg
     1021 cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa
     1081 gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg
     1141 cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg
     1201 acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctt gtttcttttc
     1261 tgtggctgcg tgaaagcctt gaggggctcc gggagggccc tttgtgcggg gggagcggct
     1321 cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc gcgctgcccg
     1381 gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt gtgcgcgagg
     1441 ggagcgcggc cgggggcggt gccccgcggt gcgggggggg ctgcgagggg aacaaaggct
     1501 gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg gtcgggctgc
     1561 aaccccccct gcacccccct ccccgagttg ctgagcacgg cccggcttcg ggtgcggggc
     1621 tccgtacggg gcgtggcgcc gggctcgccg tgccgggcgg ggggtggcgg caggtggggg
     1681 tgccgggcgg ggcggggccg cctcgggccg gggagggctc gggggagggg cgcggcggcc
     1741 cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat
     1801 cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg tgcggagccg aaatctggga
     1861 ggcgccgccg caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc ggcaggaagg
     1921 aaatgggcgg ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc cctctccagc
     1981 ctcggggctg tccgcggggg gacggctgcc ttcggggggg acggggcagg gcggggttcg
     2041 gcttctggcg tgtgaccggc ggctctagag cctctgctaa ccatgttcat gccttcttct
     2101 ttttcctaca gctcctgggc aacgtgctgg ttattgtgct gtctcatcat tttggcaaag
     2161 aattctgcag tcgacggtac cgcgggcccg ggatccaccg gtcgccacca tgggcgcccc
     2221 aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccagcgacc tggtgctggg
     2281 cctggacatc ggcatcggca gcgtgggcgt gggcatcctg aacaaggtga ccggcgagat
     2341 catccacaag aacagtcgca tcttccctgc tgctcaggct gagaacaacc tggtgcgccg
     2401 caccaaccgc cagggtcgcc ggcttgctcg ccgcaagaag caccggcgcg tgcgcctgaa
     2461 ccgcctgttc gaggagagcg gcctgatcac cgacttcacc aagatcagca tcaacctgaa
     2521 cccctaccag ctgcgcgtga agggcctgac cgacgagctg agcaacgagg agctgttcat
     2581 cgccctgaag aacatggtga agcaccgcgg catcagctac ctggacgacg ccagcgacga
     2641 cggcaacagc agcgtgggcg actacgccca gatcgtgaag gagaacagca agcagctgga
     2701 gaccaagacc cccggccaga tccagctgga gcgctaccag acctacggcc agctgcgcgg
     2761 cgacttcacc gtggagaagg acggcaagaa gcaccgcctg atcaacgtgt tccccaccag
     2821 cgcctaccgc agcgaggccc tgcgcatcct gcagacccag caggagttca acccccagat
     2881 caccgacgag ttcatcaacc gctacctgga gatcctgacc ggcaagcgca agtactacca
     2941 cggccccggc aacgagaaga gccgcaccga ctacggccgc taccgcacca gcggcgagac
     3001 cctggacaac atcttcggca tcctgatcgg caagtgcacc ttctaccccg acgagttccg
     3061 cgccgccaag gccagctaca ccgcccagga gttcaatctg ctgaacgacc tgaacaacct
     3121 gaccgtgccc accgagacca agaagctgag caaggagcag aagaaccaga tcatcaacta
     3181 cgtgaagaac gagaaggcta tgggccccgc caagctgttc aagtacatcg ccaagctgct
     3241 gagctgcgac gtggccgaca tcaagggcta ccgcatcgac aagagcggca aggccgagat
     3301 ccacaccttc gaggcctacc gcaagatgaa gaccctggag accctggaca tcgagcagat
     3361 ggaccgagag accctggaca agctggccta cgtgctgacc ctgaacaccg agcgcgaggg
     3421 catccaggag gccctggagc acgagttcgc cgacggcagc ttcagccaga aacaggtgga
     3481 cgagctggtg cagttccgca aggccaacag cagcatcttc ggcaagggct ggcacaactt
     3541 cagcgtgaag ctgatgatgg agctgatccc cgagctgtac gagaccagcg aggagcagat
     3601 gaccatcctg acccgcctgg gcaagcagaa gaccaccagc agcagcaaca agaccaagta
     3661 catcgacgag aagctgctga ccgaggagat ctacaacccc gtggtggcca agagcgtgcg
     3721 ccaggccatc aagatcgtga acgccgccat caaggagtac ggcgacttcg acaacatcgt
     3781 gatcgagatg gcccgcgaga ccaacgagga cgacgagaag aaggccatcc agaagatcca
     3841 gaaggccaac aaggacgaga aggacgccgc catgctgaag gccgccaacc agtacaacgg
     3901 caaggccgag ctgccccaca gcgtgttcca cggccacaag cagctggcca ccaagatccg
     3961 cctgtggcac cagcagggcg agcgctgcct gtacaccggc aagaccatca gcatccacga
     4021 cctgatcaac aacagcaacc agttcgaggt ggaccacatc ctgcccctga gcatcacctt
     4081 cgacgacagc ctggccaaca aggtgctggt gtacgccacc gccaaccagg agaagggcca
     4141 gcgcaccccc taccaggccc tggacagcat ggacgacgcc tggagcttcc gcgagctgaa
     4201 ggccttcgtg cgcgagagca agaccctgag caacaagaag aaggagtatc tgctgaccga
     4261 ggaggacatc agcaagttcg acgtgcgcaa gaagttcatc gagcgcaacc tggtggacac
     4321 ccgctacgcc agccgcgtgg tgctgaacgc cctgcaggag cacttccgcg cccacaagat
     4381 cgacaccaag gtgagcgtgg tgcgcggcca gttcaccagc cagctgcgcc gccactgggg
     4441 catcgagaag acccgcgaca cctaccacca ccacgccgtg gacgccctga tcattgcggc
     4501 ttctagccag ctgaacctgt ggaagaagca gaagaacacc ctggtgagct acagcgagga
     4561 ccagctgctg gacatcgaga ccggcgagct gatcagcgac gacgagtaca aggagagcgt
     4621 gttcaaggcc ccctaccagc acttcgtgga caccctgaag agcaaggagt tcgaggacag
     4681 catcctgttc agctaccagg tggacagcaa gttcaaccgc aagatcagcg acgccaccat
     4741 ctacgccacc cgccaggcca aggtgggcaa ggacaaggcc gacgagacct acgtgctggg
     4801 caagatcaag gacatctaca cccaggacgg ctacgacgcc ttcatgaaga tctacaagaa
     4861 ggacaagagc aagttcctga tgtaccgcca cgacccccag accttcgaga aggtgatcga
     4921 gcccatcctg gagaactacc ccaacaagca gatcaacgag aaaggcaagg aggtgccctg
     4981 caaccccttc ctgaagtaca aggaggagca cggctacatc cgcaagtaca gcaagaaggg
     5041 caacggcccc gagatcaaga gcctgaagta ctacgacagc aagctgggca accacatcga
     5101 catcaccccc aaggacagca acaacaaggt ggtgctgcag agccttaagc cctggcgcac
     5161 cgacgtgtac ttcaacaaga acaccggcaa gtacgagatc ctggggctga agtacagcga
     5221 tatgcagttt gagaaaggca caggcaagta cagcatcagc aaggagcagt acgagaacat
     5281 caaggtgcgc gagggcgtgg acgagaacag cgagttcaag ttcaccctgt acaagaacga
     5341 ccttctgctg ctgaaggaca gcgagaacgg cgagcaaatc ctgctgcgct tcaccagccg
     5401 caacgacacc agcaagcact acgtggagct gaagccctac aaccgccaga agttcgaggg
     5461 cagcgagtac ctgatcaaga gcctgggcac cgtggtgaag ggcggcaggt gcatcaaggg
     5521 cctgggcaag agcaacatca gcatctacaa ggtgcgcacc gacgtgctgg gcaaccagca
     5581 catcatcaag aacgagggcg acaagcccaa gttggacttc agcagggctg accccaagaa
     5641 gaagaggaag gtgggatcct gataagaatt cgcggccgca ctcctcaggt gcaggctgcc
     5701 tatcagaagg tggtggctgg tgtggccaat gccctggctc acaaatacca ctgagatctt
     5761 tttccctctg ccaaaaatta tggggacatc atgaagcccc ttgagcatct gacttctggc
     5821 taataaagga aatttatttt cattgcaata gtgtgttgga attttttgtg tctctcactc
     5881 ggaaggacat atgggagggc aaatcattta aaacatcaga atgagtattt ggtttagagt
     5941 ttggcaacat atgcccatat gctggctgcc atgaacaaag gttggctata aagaggtcat
     6001 cagtatatga aacagccccc tgctgtccat tccttattcc atagaaaagc cttgacttga
     6061 ggttagattt tttttatatt ttgttttgtg ttattttttt ctttaacatc cctaaaattt
     6121 tccttacatg ttttactagc cagatttttc ctcctctcct gactactccc agtcatagct
     6181 gtccctcttc tcttatggag atccctcgac ctgcagccca agcttggcgt aatcatggtc
     6241 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     6301 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     6361 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagcggatcc gcatctcaat
     6421 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     6481 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     6541 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     6601 tgcaaaaagc taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     6661 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     6721 tcaatgtatc ttatcatgtc tggatccgct gcattaatga atcggccaac gcgcggggag
     6781 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     6841 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     6901 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     6961 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     7021 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     7081 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     7141 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     7201 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     7261 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     7321 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     7381 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     7441 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     7501 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     7561 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     7621 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     7681 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     7741 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     7801 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     7861 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     7921 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     7981 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     8041 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     8101 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     8161 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     8221 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     8281 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     8341 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     8401 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     8461 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     8521 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     8581 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     8641 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     8701 ggttccgcgc acatttcccc gaaaagtgcc acctg
//
