LOCUS       sequence-328001-        7449 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SlutrCas9, and cloning backbone for sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7449)
  AUTHORS   Hu Z, Zhang C, Wang S, Gao S, Wei J, Li M, Hou L, Mao H, Wei Y, Qi 
            T, Liu H, Liu D, Lan F, Lu D, Wang H, Li J, Wang Y
  TITLE     Discovery and engineering of small SlugCas9 with broad targeting 
            range and high specificity and activity.
  JOURNAL   Nucleic Acids Res. 2021 Mar 15. pii: 6171296. doi: 
            10.1093/nar/gkab148.
  PUBMED    33721016
REFERENCE   2  (bases 1 to 7449)
  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: "Nucleic 
            Acids Res. 2021 Mar 15. pii: 6171296. doi: 10.1093/nar/gkab148."
COMMENT     Sequence Label: SlutrCas9
FEATURES             Location/Qualifiers
     source          1..7449
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      603..612
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             615..635
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3819..3866
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3873..3953
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(3981..3998)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3987..4194
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4201..4449
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4201..4221
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4372..4391
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4470..4545
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4559..4699
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4559..4688
                     /label=AAV2 ITR
     rep_origin      4774..5229
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4861..4880)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5071..5092
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5246..5265)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5365..5387
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5425..5443)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5511..5615
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5616..5684,5685..6476)
                     /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     complement(5834..5853)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6647..7235
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7136..7155
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   7297..7426
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactaccg
      601 gtgccaccat ggccccaaag aagaagcgga aggtcggtat ccacggagtc ccagcagcca
      661 gaaacagcta catcctgggc ctggacatcg gaatcaccag cgtgggatat ggcatcatcg
      721 acagagtcac cagagaggtg atcgacgccg gcgtgcggct tttccccgag gccaacgtgg
      781 agaacaacga gggcagacgg agcaagagag gagcccggcg gctgaaaaga aggcggatcc
      841 accggctgaa tagaatcaag cagctgctga aaaacgccgg cctgctggag ggagatgtgc
      901 tgcctaagtc taccaacccc tatgagatca gagtgcgggg cctccgaagc cctctgacca
      961 aagatgaact ggtgatcgcc ctgctccaca tcgccaaaag aagaggcatc cacaacatca
     1021 acatcgtggg agatgacgaa gaaacggaca gcacactgag taccacagcc cagctgaaga
     1081 agaacgagaa ggctctcaag ggacagtttg tttgtgaact gcaactggac agactggcta
     1141 atgcccacca agtgcggggc gagaaaaatc gatttaagac agaggacatt gtgaaggaag
     1201 tcagagccct gcttcagcaa cagcagaact tccacaacat cgataattct ttcgtggaac
     1261 agtacattgc cctgctggag agccggagga cctactacga gggccctggc gaaggctctc
     1321 cttacggctg ggacggcgac attaagaagt ggtacgagat gctgatgggc tattgcacct
     1381 acttccctga agagctgaga agcgtgaagt acgcctacac cgccgatctg ttcaacgccc
     1441 tgaatgacct gaacaacctg gtgatcaccc gggacgacaa cagcaaattg acatacgccg
     1501 agaagtacca tatcatcgag aacgtgttca aacagaagaa agtacctaca ctgaagcaga
     1561 tcgccaagga aatcggagtg aacgagagcg atattaaggg ctacagaatc aacaaatctg
     1621 agaaacctct gttcaccagc ttcaaactgt accacgatct gaagagcgtg ttcagcgacc
     1681 ctacaaaact ggaagatatc gacttgctgg accgaatcgc cgtggtgctg accatgtacc
     1741 aagatgccga atccatgaaa gccgccctga acaccttccc tgaagtgttc agcgaagcag
     1801 agaaagagaa gctgagcgcc ctcacaggct acgctggcac ccatagactg tctctgaagt
     1861 gcatgaacct gctgatccct gatctgtggc agacaagcct gaaccagatg gaactgttcg
     1921 tgaagctgaa tctgaaacca cagaagctgg acctgagcca gtgccaccag attcctaccc
     1981 agctggtgga cgagttcatc ctgtctcctg tggtgaaaag agccttcacc caaagcatca
     2041 aggtgatcaa cgccatcatc cagaaatacg gcctgcccga cgacatcata atcgagctgg
     2101 ccagggaaaa aaacagcgcc gataagcgga agttcctgaa tcagctgcag aagaagaacg
     2161 agaaggcccg gcacgagatc aataccctgg tggcccagta cggccagcca aatgctaagc
     2221 ggctggtgga aaagatcaca ctgcaccagc agcaggaggg caagtgtctg tactccctga
     2281 aggatatccc cctggagcag ctgctgaagc agccctacct gtacgaggtg gaccacatca
     2341 tccccagaag cgtttctttc gacaacagca tgcagaacaa ggtgctggtc ctggccgaag
     2401 aaaacgccaa aaagggcaac cagacccctt accagtacct gaatagcaga gaggccagca
     2461 tgacctaccc cgaattcaaa cagcacatcc tgaatctgag caaggccaag gaccggatca
     2521 gcaagaagaa gcggaactac ctgctcgagg aaagagatat caacaagttc gacgtgcaaa
     2581 aggacttcat caacagaaac ctggttgaca ccagatacgc caccagagag ctcgcctctc
     2641 tgctgaaggc ttatttcaag acacatgagc tccctgtgaa agtgaagacc atcaacggcg
     2701 gatttaccca ctacctgaga aaggtgtgga agtttgacaa agatagaaac aagggctaca
     2761 agcaccacgc cgaggatgca ctgatcatcg ccaacgccga ctttctgttt aagaaccaga
     2821 ctctgaacaa aatcgaggct atcctgaacg agcccggcag agaggtggaa tctgacacag
     2881 tgaaggtgca gagcgaggac aattaccagg acctgtttga gaacaccaag aaggctttcg
     2941 ccatcaagaa tttcaaggat ttcaagttta gccacagagt ggaccagaag cctaaccggc
     3001 agctcgtgaa cgacaccctg tacagcacca gagaggtgaa cgaagatctg tacgtggtgc
     3061 agaccctgaa ggacatctac agcaaggaca acaaagacgt gaagcggctc ttcgacaagc
     3121 aacccgagaa gttcctgatg ttccagcacg accccgagac attcaagaaa ttcgagctgg
     3181 ctatgaagca atatgccgaa gagaagaacc ctctggctag atactacgag gaacagggct
     3241 acatcaccaa gtacgccaag aaaggagacg gcccacctgt gaaaagcctc aaatacatcg
     3301 gcaagaaggt gggaaaacac ctggatgtga ccggcgacta cgaggatagc aaccggaagc
     3361 tggtgaagct gagcctgaag tcctttagat tcgatatcta ccacacagac aagggctaca
     3421 agatggttcc aatcacttac ctggatgtgc agaaaaaaga gaagtactac tacatcccca
     3481 ccgagaaata cgaagccctg aagcaggaga agggcatcaa ccagaatgct cagttcattg
     3541 gcagcttcta ctacaacgac ctgatcgagt tcgatggcga gctgtataga gtgatcggca
     3601 tcaacaacgg cgacaagaat ctcgttgaac tcgacatggt ggacattaga tataaggaat
     3661 actgcgagct gaactccatc accaccacac ctagaatcgt taagaccatc agccccaaga
     3721 cccagagcat cgagaagtac acaacagata tcctgggcaa cctgtacaaa gcccagcctg
     3781 gcaagaagcc tcagttcatc ttcaacaaag acgaggataa aaggccggcg gccacgaaaa
     3841 aggccggcca ggcaaaaaag aaaaagggat cctacccata cgatgttcca gattacgctt
     3901 acccatacga tgttccagat tacgcttacc catacgatgt tccagattac gcttaagaat
     3961 tcctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     4021 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     4081 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     4141 ggggcaggac agcaaggggg aggattggga agagaatagc aggcatgctg gggaggtacc
     4201 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
     4261 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
     4321 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
     4381 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
     4441 cgaaacaccg gagaccacgg caggtctcag ttttagtact ctggaaacag aatctactaa
     4501 aacaaggcaa aatgccgtgt ttatctcgtc aacttgttgg cgagattttt gcggccgcag
     4561 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc
     4621 gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga
     4681 gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     4741 atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg
     4801 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg
     4861 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     4921 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     4981 aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     5041 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     5101 tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt
     5161 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt
     5221 ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
     5281 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     5341 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     5401 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     5461 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     5521 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     5581 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     5641 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     5701 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     5761 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     5821 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     5881 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     5941 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     6001 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     6061 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     6121 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     6181 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     6241 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     6301 ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca gcactggggc
     6361 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     6421 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     6481 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     6541 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     6601 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     6661 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     6721 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     6781 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     6841 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     6901 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     6961 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     7021 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     7081 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     7141 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     7201 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     7261 ggttcctggc cttttgctgg ccttttgctc acatgtcctg caggcagctg cgcgctcgct
     7321 cgctcactga ggccgcccgg gcgtcgggcg acctttggtc gcccggcctc agtgagcgag
     7381 cgagcgcgca gagagggagt ggccaactcc atcactaggg gttcctgcgg cctctagact
     7441 cgaggcgtt
//
