LOCUS       sequence-317660-        7450 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses highly specific SlugCas9, and cloning backbone for sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7450)
  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 7450)
  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: SlugCas9-HF
FEATURES             Location/Qualifiers
     source          1..7450
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        154..533
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        534..737
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     688..708
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      756..765
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             768..788
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3972..4019
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4026..4106
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(4134..4151)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4140..4347
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4355..4603
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4355..4375
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4526..4545
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4624..4699
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4713..4853
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4713..4842
                     /label=AAV2 ITR
     rep_origin      4928..5383
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5015..5034)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5225..5246
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5400..5419)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5519..5541
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5579..5597)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5665..5769
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5770..5838,5839..6630)
                     /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(5988..6007)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6801..7389
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7290..7309
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcctcta gactcgaggc gttgacattg attattgact agttattaat
      181 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
      241 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
      301 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      361 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      421 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
      481 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
      541 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
      601 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
      661 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
      721 tctatataag cagagctctc tggctaacta ccggtgccac catggcccca aagaagaagc
      781 ggaaggtcgg tatccacgga gtcccagcag ccaaccaaaa attcatactg ggactggaca
      841 tcggaatcac cagcgtgggc tacggcctga tcgactacga gacaaagaat atcatcgatg
      901 ccggcgttag actgttcccc gaggccaacg tggaaaacaa cgagggaaga aggtccaaac
      961 gtggaagcag aagactgaag cgacgccgca ttcacagact tgaacgggtg aagaagctgc
     1021 tcgaggatta taatctgctg gatcagtccc agattcctca gtctacaaac ccctacgcca
     1081 tccgcgtgaa gggcctgtct gaagccctga gcaaggacga actcgtgatt gccctgctcc
     1141 atatcgccaa gagaagaggc atccacaaga tcgacgtgat cgacagcaac gacgacgtgg
     1201 ggaacgagct cagcaccaag gaacagctga ataagaacag caagctgctg aaagacaaat
     1261 ttgtgtgcca gatccagctg gaaagaatga atgagggcca ggtgcgggga gagaaaaacc
     1321 ggttcaagac cgctgatatc atcaaggaaa tcatccagct gctgaatgtg cagaagaact
     1381 tccaccagct ggacgagaac ttcatcaaca agtacatcga actggttgag atgagacggg
     1441 aatacttcga gggccccggc aagggcagtc catatggctg ggaaggcgac cctaaggctt
     1501 ggtacgagac actgatgggc cactgcacct acttcccaga tgagctggct agcgtgaaat
     1561 acgcctacag tgccgacctg ttcaacgctc tgaacgacct gaacaacctg gtcatccaaa
     1621 gagatggact gtctaagctc gagtatcatg agaagtatca catcatcgag aacgtgttca
     1681 agcagaagaa gaaacctaca ctgaagcaga tcgccaatga gatcaatgtc aaccctgaag
     1741 atatcaaggg ctacagaatc acaaagtctg gcaagcccca gtttaccgag tttaagctct
     1801 accacgacct gaaaagcgtg ctgtttgacc agagcatcct ggagaacgaa gacgtgctgg
     1861 accagatcgc tgagatcctg accatctacc aggacaagga tagcatcaaa tctaagctga
     1921 cggaactgga catcctgctg aacgaggaag ataaggaaaa catcgcccag ctgactggct
     1981 acaccgggac ccaccggctc agcctgaaat gcatccggct ggtcctggaa gagcagtggt
     2041 attctagccg ggctcagatg gaaatcttcg cccacctgaa cattaagcct aagaagatca
     2101 acctgacagc cgccaacaag atcccgaagg ctatgatcga cgagttcatc ctgagccctg
     2161 tggtgaagag gaccttcggc caggccatta accttattaa caagatcata gaaaagtacg
     2221 gcgtgcctga agatatcatc atcgagctgg ccagagaaaa taatagcaag gacaagcaga
     2281 agttcatcaa tgagatgcag aaaaagaacg agaacaccag aaagagaatt aacgaaatca
     2341 tcggcaagta tggcaaccag aacgccaaga gactggtcga gaagattaga ctgcacgacg
     2401 agcaggaggg caagtgcctg tactcactgg aaagcatccc tctggaggac ctgctgaaca
     2461 accccaacca ctacgaggtg gaccacatca ttccaagatc tgtgtccttc gacaactctt
     2521 accacaacaa agtgctcgtg aagcagagcg agaactccaa aaaatccaac ctgacccctt
     2581 accagtactt taacagcggc aagtccaagc tctcttacaa ccagtttaaa caacacatcc
     2641 tgaacctgag caagtcccag gatagaatca gcaaaaaaaa gaaagagtat ctgctggaag
     2701 aacgggacat caacaagttc gaggtgcaaa aagagttcat caatagaaac ctggtggata
     2761 cccggtacgc cacagccgag ctgacaaact acctgaaggc ctacttcagc gccaacaata
     2821 tgaacgtgaa ggtgaaaacg atcaacggca gcttcaccga ttacctgcgg aaagtgtgga
     2881 agtttaagaa ggaacggaac cacggctaca agcaccacgc cgaggacgcc ctgattatcg
     2941 ctaatgccga tttcctgttc aaagagaaca agaagctgaa agccgtgaac tctgtgctgg
     3001 aaaaacctga gatcgagagc aagcagctgg atatccaggt ggatagcgag gataactaca
     3061 gcgaaatgtt catcatccct aagcaggtcc aggacatcaa ggacttcaga aacttcaagt
     3121 acagccacag agtggacaag aagcctaaca gacagctgat caacgataca ctgtacagca
     3181 cccggaagaa ggacaactcc acctacatcg tgcagaccat caaagatatc tatgccaaag
     3241 ataataccac cctgaagaag cagtttgaca agtcacccga gaagttcctc atgtaccaac
     3301 acgatccgcg gaccttcgag aagttggaag tgatcatgaa gcagtacgct aatgagaaga
     3361 atcctctggc caagtaccac gaggaaacag gcgagtacct gaccaaatac agcaaaaaaa
     3421 acaacggccc tatcgtgaaa agcctgaagt acattggaaa caagctgggc agccacctag
     3481 atgtgaccca ccagttcaag agcagcacca agaagttggt gaagctgagc atcaagcctt
     3541 atagattcga cgtctacctg accgacaagg gatataagtt catcaccatc agctacctgg
     3601 acgtgctgaa gaaagacaat tactactaca tacccgaaca gaagtacgac aagctcaaac
     3661 tgggcaaggc catcgacaaa aacgccaagt ttatcgctag cttctacaag aatgatctga
     3721 tcaagctgga cggcgagatc tacaagatca tcggcgtgaa tagcgacacc agaaacatga
     3781 tcgaactgga tctgcctgac atcagataca aagaatactg cgagctgaac aatatcaagg
     3841 gcgaacctag aatcaaaaag accatcggca aaaaggtgaa tagcatcgaa aaactgacaa
     3901 ccgacgtgct gggcaacgtg ttcaccaaca cccagtacac aaaacctcag ctgctgttca
     3961 agcgaggaaa taaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagg
     4021 gatcctaccc atacgatgtt ccagattacg cttacccata cgatgttcca gattacgctt
     4081 acccatacga tgttccagat tacgcttaag aattcctaga gctcgctgat cagcctcgac
     4141 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     4201 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     4261 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     4321 ggaagagaat agcaggcatg ctggggaggt acctgagggc ctatttccca tgattccttc
     4381 atatttgcat atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa
     4441 cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc
     4501 agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt
     4561 cgatttcttg gctttatata tcttgtggaa aggacgaaac accggagacc acggcaggtc
     4621 tcagttttag tactctggaa acagaatcta ctaaaacaag gcaaaatgcc gtgtttatct
     4681 cgtcaacttg ttggcgagat ttttgcggcc gcaggaaccc ctagtgatgg agttggccac
     4741 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
     4801 gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct
     4861 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa
     4921 ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     4981 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     5041 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     5101 cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt
     5161 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     5221 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggg ctattctttt
     5281 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     5341 aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt
     5401 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
     5461 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     5521 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc
     5581 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     5641 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     5701 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     5761 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     5821 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     5881 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     5941 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     6001 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     6061 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     6121 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     6181 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     6241 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     6301 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     6361 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     6421 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     6481 cgtggaagcc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     6541 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     6601 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     6661 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     6721 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     6781 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     6841 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     6901 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     6961 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     7021 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     7081 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     7141 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     7201 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     7261 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     7321 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     7381 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     7441 gctcacatgt
//
