LOCUS       sequence-273359-        9320 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses St1BE4max-TH1477 in mammalian cells along with its 
            U6-driven sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9320)
  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 9320)
  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_APOBEC-1_hSt1Cas9_TH1477_2xUGI_3xHA
FEATURES             Location/Qualifiers
     source          1..9320
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        40..288
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     40..60
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     211..230
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        460..763
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          1041..2057
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1981..2003
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2065..2084
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2160..2180
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2181..2864
                     /codon_start=1
                     /product="cytidine deaminase (C to U editing enzyme) from 
                     rat"
                     /label=APOBEC-1
                     /note="can use ssDNA as a substrate (Komor et al., 2016)"
                     /translation="SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGG
                     RHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLS
                     RYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNE
                     AHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHIL
                     WATGLK"
     CDS             6330..6350
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6387..6635
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             6666..6914
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             6957..6977
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6984..7064
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     polyA_signal    7106..7330
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(7403..7420)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7574..8162)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7654..7673)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8333..9124,9125..9193))
                     /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     8956..8975
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9194..9298)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cctgggtcga ctggatccgg taccaaggtc gggcaggaag agggcctatt tcccatgatt
       61 ccttcatatt tgcatatacg atacaaggct gttagagaga taattagaat taatttgact
      121 gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag
      181 tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag
      241 tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacacccg agacgattaa
      301 tgcgtctcgg tctttgtact ctggtaccag aagctacaaa gataaggctt catgccgaaa
      361 tcaacaccct gtcattttat ggcagggtgt tttttttact agttattaat agtaatcaat
      421 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
      481 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
      541 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
      601 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
      661 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
      721 tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac
      781 gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat
      841 tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggsgcgcg ccrggsgggg
      901 sggggsgggg sggggggsgg ggsggggsgr ggcggagagg tgcggcggca gccaatcaga
      961 gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa
     1021 agcgaagcgc gcggcgggcg ggagtcgctg cgcgctgcct tcgccccgtg ccccgctccg
     1081 ccgccgcctc gcgccgcccg ccccggctct gactgaccgc gttactccca caggtgagcg
     1141 ggcgggacgg cccttctcct ccgggctgta attagcgctt ggtttaatga cggcttgttt
     1201 cttttctgtg gctgcgtgaa agccttgagg ggctccggga gggccctttg tgcgggggga
     1261 gcggctcggg gggtgcgtgc gtgtgtgtgt gcgtggggag cgccgcgtgc ggctccgcgc
     1321 tgcccggcgg ctgtgagcgc tgcgggcgcg gcgcggggct ttgtgcgctc cgcagtgtgc
     1381 gcgaggggag cgcggccggg ggcggtgccc cgcggtgcgg ggggggctgc gaggggaaca
     1441 aaggctgcgt gcggggtgtg tgcgtggggg ggtgagcagg gggtgtgggc gcgtcggtcg
     1501 ggctgcaacc ccccctgcac ccccctcccc gagttgctga gcacggcccg gcttcgggtg
     1561 cggggctccg tacggggcgt ggcgcggggc tcgccgtgcc gggcgggggg tggcggcagg
     1621 tgggggtgcc gggcggggcg gggccgcctc gggccggggr gggctcgggg gaggggcgcg
     1681 gcggcccccg gagcgccggc ggctgtcgag gcgcggcgag ccgcagccat tgccttttat
     1741 ggtaatcgtg cgagagggcg cagggacttc ctttgtccca aatctgtgcg gagccgaaat
     1801 ctgggaggcg ccgccgcacc ccctctagcg ggcgcggggc gaagcggtgc ggcgccggca
     1861 ggaaggaaat gggcggggag ggccttcgtg cgtcgccgcg ccgccgtccc cttctccctc
     1921 tccagcctcg gggctgtccg cggggggacg gctgccttcg ggggggacgg ggcagggcgg
     1981 ggttcggctt ctggcgtgtg accggcggct ctagagcctc tgctaaccat gttcatgcct
     2041 tcttcttttt cctacagctc ctgggcaacg tgctggttat tgtgctgtct catcattttg
     2101 gcaaagaatt cagagccgcc accatgaaac ggacagccga cggaagcgag ttcgagtcac
     2161 caaagaagaa gcggaaagtc tcctcagaga ctgggcctgt cgccgtcgat ccaaccctgc
     2221 gccgccggat tgaacctcac gagtttgaag tgttctttga cccccgggag ctgagaaagg
     2281 agacatgcct gctgtacgag atcaactggg gaggcaggca ctccatctgg aggcacacct
     2341 ctcagaacac aaataagcac gtggaggtga acttcatcga gaagtttacc acagagcggt
     2401 acttctgccc caataccaga tgtagcatca catggtttct gagctggtcc ccttgcggag
     2461 agtgtagcag ggccatcacc gagttcctgt ccagatatcc acacgtgaca ctgtttatct
     2521 acatcgccag gctgtatcac cacgcagacc caaggaatag gcagggcctg cgcgatctga
     2581 tcagctccgg cgtgaccatc cagatcatga cagagcagga gtccggctac tgctggcgga
     2641 acttcgtgaa ttattctcct agcaacgagg cccactggcc taggtaccca cacctgtggg
     2701 tgcgcctgta cgtgctggag ctgtattgca tcatcctggg cctgccccct tgtctgaata
     2761 tcctgcggag aaagcagccc cagctgacct tctttacaat cgccctgcag tcttgtcact
     2821 atcagaggct gccaccccac atcctgtggg ccacaggcct gaagtctgga ggatctagcg
     2881 gaggctcctc tggcagcgag acaccaggaa caagcgagtc agcaacacca gagagcagtg
     2941 gcggcagcag cggcggcagc agcgacctgg tgctgggcct ggctatcggc atcggcagcg
     3001 tgggcgtggg catcctgaac aaggtgaccg gcgagatcat ccacaagaac agtcgcatct
     3061 tccctgctgc tcaggctgag aacaacctgg tgcgccgcac caaccgccag ggtcgccggc
     3121 ttgctcgccg caagaagcac cggcgcgtgc gcctgaaccg cctgttcgag gagagcggcc
     3181 tgatcaccga cttcaccaag atcagcatca acctgaaccc ctaccagctg cgcgtgaagg
     3241 gcctgaccga cgagctgagc aacgaggagc tgttcatcgc cctgaagaac atggtgaagc
     3301 accgcggcat cagctacctg gacgacgcca gcgacgacgg caacagcagc gtgggcgact
     3361 acgcccagat cgtgaaggag aacagcaagc agctggagac caagaccccc ggccagatcc
     3421 agctggagcg ctaccagacc tacggccagc tgcgcggcga cttcaccgtg gagaaggacg
     3481 gcaagaagca ccgcctgatc aacgtgttcc ccaccagcgc ctaccgcagc gaggccctgc
     3541 gcatcctgca gacccagcag gagttcaacc cccagatcac cgacgagttc atcaaccgct
     3601 acctggagat cctgaccggc aagcgcaagt actaccacgg ccccggcaac gagaagagcc
     3661 gcaccgacta cggccgctac cgcaccagcg gcgagaccct ggacaacatc ttcggcatcc
     3721 tgatcggcaa gtgcaccttc taccccgacg agttccgcgc cgccaaggcc agctacaccg
     3781 cccaggagtt caatctgctg aacgacctga acaacctgac cgtgcccacc gagaccaaga
     3841 agctgagcaa ggagcagaag aaccagatca tcaactacgt gaagaacgag aaggctatgg
     3901 gccccgccaa gctgttcaag tacatcgcca agctgctgag ctgcgacgtg gccgacatca
     3961 agggctaccg catcgacaag agcggcaagg ccgagatcca caccttcgag gcctaccgca
     4021 agatgaagac cctggagacc ctggacatcg agcagatgga ccgagagacc ctggacaagc
     4081 tggcctacgt gctgaccctg aacaccgagc gcgagggcat ccaggaggcc ctggagcacg
     4141 agttcgccga cggcagcttc agccagaaac aggtggacga gctggtgcag ttccgcaagg
     4201 ccaacagcag catcttcggc aagggctggc acaacttcag cgtgaagctg atgatggagc
     4261 tgatccccga gctgtacgag accagcgagg agcagatgac catcctgacc cgcctgggca
     4321 agcagaagac caccagcagc agcaacaaga ccaagtacat cgacgagaag ctgctgaccg
     4381 aggagatcta caaccccgtg gtggccaaga gcgtgcgcca ggccatcaag atcgtgaacg
     4441 ccgccatcaa ggagtacggc gacttcgaca acatcgtgat cgagatggcc cgcgagacca
     4501 acgaggacga cgagaagaag gccatccaga agatccagaa ggccaacaag gacgagaagg
     4561 acgccgccat gctgaaggcc gccaaccagt acaacggcaa ggccgagctg ccccacagcg
     4621 tgttccacgg ccacaagcag ctggccacca agatccgcct gtggcaccag cagggcgagc
     4681 gctgcctgta caccggcaag accatcagca tccacgacct gatcaacaac agcaaccagt
     4741 tcgaggtgga ccacatcctg cccctgagca tcaccttcga cgacagcctg gccaacaagg
     4801 tgctggtgta cgccaccgcc aaccaggaga agggccagcg caccccctac caggccctgg
     4861 acagcatgga cgacgcctgg agcttccgcg agctgaaggc cttcgtgcgc gagagcaaga
     4921 ccctgagcaa caagaagaag gagtatctgc tgaccgagga ggacatcagc aagttcgacg
     4981 tgcgcaagaa gttcatcgag cgcaacctgg tggacacccg ctacgccagc cgcgtggtgc
     5041 tgaacgccct gcaggagcac ttccgcgccc acaagatcga caccaaggtg agcgtggtgc
     5101 gcggccagtt caccagccag ctgcgccgcc actggggcat cgagaagacc cgcgacacct
     5161 accaccacca cgccgtggac gccctgatca ttgcggcttc tagccagctg aacctgtgga
     5221 agaagcagaa gaacaccctg gtgagctaca gcgaggacca gctgctggac atcgagaccg
     5281 gcgagctgat cagcgacgac gagtacaagg agagcgtgtt caaggccccc taccagcact
     5341 tcgtggacac cctgaagagc aaggagttcg aggacagcat cctgttcagc taccaggtgg
     5401 acagcaagtt caaccgcaag atcagcgacg ccaccatcta cgccacccgc caggccaagg
     5461 tgggcaagga caaggccgac gagacctacg tgctgggcaa gatcaaggac atctacaccc
     5521 aggacggcta cgacgccttc atgaagatct acaagaagga caagagcaag ttcctgatgt
     5581 accgccacga cccccagacc ttcgagaagg tgatcgagcc catcctggag aactacccca
     5641 acaagcagat caacgagaaa ggcaaggagg tgccctgcaa ccccttcctg aagtacaagg
     5701 aggagcacgg ctacatccgc aagtacagca agaagggcaa cggccccgag atcaagagcc
     5761 tgaagtacta cgacagcaag ctgggcaacc acatcgacat cacccccaag gacagcaaca
     5821 acaaggtggt gctgcagagc gtgagcccct ggcgcgccga cgtgtacttc aacaagaaca
     5881 ccggcaagta cgagatcctg gggctgaagt acagcgatat gcagtttgag aaaggcacag
     5941 gcaagtacag catcagcaag gagcagtacg agaacatcaa ggtgcgcgag ggcgtggacg
     6001 agaacagcga gttcaagttc accctgtaca agaacgacct tctgctgctg aaggacagcg
     6061 agaacggcga gcaaatcctg ctgcgcttca ccagccgcaa cgacaccagc aagcactacg
     6121 tggagctgaa gccctacaac cgccagaagt tcgagggcag cgagtacctg atcaagagcc
     6181 tgggcaccgt ggtgaagggc ggcaggtgca tcaagggcct gggcaagagc aacatcagca
     6241 tctacaaggt gcgcaccgac gtgctgggca accagcacat catcaagaac gagggcgaca
     6301 agcccaagtt ggacttcagc agggctgacc ccaagaagaa gaggaaggtg ggatctagcg
     6361 gcgggagcgg cgggagcggg gggagcacta atctgagcga catcattgag aaggagactg
     6421 ggaaacagct ggtcattcag gagtccatcc tgatgctgcc tgaggaggtg gaggaagtga
     6481 tcggcaacaa gccagagtct gacatcctgg tgcacaccgc ctacgacgag tccacagatg
     6541 agaatgtgat gctgctgacc tctgacgccc ccgagtataa gccttgggcc ctggtcatcc
     6601 aggattctaa cggcgagaat aagatcaaga tgctgagcgg aggatccgga ggatctggag
     6661 gcagcaccaa cctgtctgac atcatcgaga aggagacagg caagcagctg gtcatccagg
     6721 agagcatcct gatgctgccc gaagaagtcg aagaagtgat cggaaacaag cctgagagcg
     6781 atatcctggt ccataccgcc tacgacgaga gtaccgacga aaatgtgatg ctgctgacat
     6841 ccgacgcccc agagtataag ccctgggctc tggtcatcca ggattccaac ggagagaaca
     6901 aaatcaaaat gctgtctggc ggctcaaaaa gaaccgccga cggcagcgaa ttcgagccca
     6961 agaagaagag gaaagtcgga tcctacccat acgatgttcc agattacgct tatccctacg
     7021 acgtgcctga ttatgcatac ccatatgatg tccccgacta tgcctaattc gaaaagtgat
     7081 aactcgagtc tagaaaccct gcaggctgtg ccttctagtt gccagccatc tgttgtttgc
     7141 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     7201 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     7261 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     7321 ggctctatgg agatccgctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     7381 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     7441 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     7501 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     7561 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     7621 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     7681 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     7741 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     7801 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     7861 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     7921 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     7981 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     8041 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     8101 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     8161 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     8221 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     8281 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     8341 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     8401 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     8461 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     8521 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     8581 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     8641 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     8701 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     8761 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     8821 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     8881 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     8941 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     9001 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     9061 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     9121 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     9181 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     9241 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     9301 catttccccg aaaagtgcca
//
