LOCUS       sequence-328009-        7449 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SlugCas9, 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: SlugCas9
FEATURES             Location/Qualifiers
     source          1..7449
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        126..505
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        506..709
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     660..680
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      728..737
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             740..760
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3944..3991
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3998..4078
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(4106..4123)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4112..4319
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4326..4574
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4326..4346
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4497..4516
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4595..4670
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4684..4824
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4684..4813
                     /label=AAV2 ITR
     rep_origin      4899..5354
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4986..5005)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5196..5217
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5371..5390)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5490..5512
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5550..5568)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5636..5740
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5741..5809,5810..6601)
                     /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(5959..5978)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6772..7360
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7261..7280
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(7422..7449,1..102)
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 cactgaggcc gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag
       61 cgcgcagaga gggagtggcc aactccatca ctaggggttc ctgcggcctc tagactcgag
      121 gcgttgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca
      181 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
      241 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
      301 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
      361 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
      421 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
      481 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg
      541 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt
      601 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac
      661 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc tctggctaac
      721 taccggtgcc accatggccc caaagaagaa gcggaaggtc ggtatccacg gagtcccagc
      781 agccaaccaa aaattcatac tgggactgga catcggaatc accagcgtgg gctacggcct
      841 gatcgactac gagacaaaga atatcatcga tgccggcgtt agactgttcc ccgaggccaa
      901 cgtggaaaac aacgagggaa gaaggtccaa acgtggaagc agaagactga agcgacgccg
      961 cattcacaga cttgaacggg tgaagaagct gctcgaggat tataatctgc tggatcagtc
     1021 ccagattcct cagtctacaa acccctacgc catccgcgtg aagggcctgt ctgaagccct
     1081 gagcaaggac gaactcgtga ttgccctgct ccatatcgcc aagagaagag gcatccacaa
     1141 gatcgacgtg atcgacagca acgacgacgt ggggaacgag ctcagcacca aggaacagct
     1201 gaataagaac agcaagctgc tgaaagacaa atttgtgtgc cagatccagc tggaaagaat
     1261 gaatgagggc caggtgcggg gagagaaaaa ccggttcaag accgctgata tcatcaagga
     1321 aatcatccag ctgctgaatg tgcagaagaa cttccaccag ctggacgaga acttcatcaa
     1381 caagtacatc gaactggttg agatgagacg ggaatacttc gagggccccg gcaagggcag
     1441 tccatatggc tgggaaggcg accctaaggc ttggtacgag acactgatgg gccactgcac
     1501 ctacttccca gatgagctga gaagcgtgaa atacgcctac agtgccgacc tgttcaacgc
     1561 tctgaacgac ctgaacaacc tggtcatcca aagagatgga ctgtctaagc tcgagtatca
     1621 tgagaagtat cacatcatcg agaacgtgtt caagcagaag aagaaaccta cactgaagca
     1681 gatcgccaat gagatcaatg tcaaccctga agatatcaag ggctacagaa tcacaaagtc
     1741 tggcaagccc cagtttaccg agtttaagct ctaccacgac ctgaaaagcg tgctgtttga
     1801 ccagagcatc ctggagaacg aagacgtgct ggaccagatc gctgagatcc tgaccatcta
     1861 ccaggacaag gatagcatca aatctaagct gacggaactg gacatcctgc tgaacgagga
     1921 agataaggaa aacatcgccc agctgactgg ctacaccggg acccaccggc tcagcctgaa
     1981 atgcatccgg ctggtcctgg aagagcagtg gtattctagc cggaatcaga tggaaatctt
     2041 cacacacctg aacattaagc ctaagaagat caacctgaca gccgccaaca agatcccgaa
     2101 ggctatgatc gacgagttca tcctgagccc tgtggtgaag aggaccttcg gccaggccat
     2161 taaccttatt aacaagatca tagaaaagta cggcgtgcct gaagatatca tcatcgagct
     2221 ggccagagaa aataatagca aggacaagca gaagttcatc aatgagatgc agaaaaagaa
     2281 cgagaacacc agaaagagaa ttaacgaaat catcggcaag tatggcaacc agaacgccaa
     2341 gagactggtc gagaagatta gactgcacga cgagcaggag ggcaagtgcc tgtactcact
     2401 ggaaagcatc cctctggagg acctgctgaa caaccccaac cactacgagg tggaccacat
     2461 cattccaaga tctgtgtcct tcgacaactc ttaccacaac aaagtgctcg tgaagcagag
     2521 cgagaactcc aaaaaatcca acctgacccc ttaccagtac tttaacagcg gcaagtccaa
     2581 gctctcttac aaccagttta aacaacacat cctgaacctg agcaagtccc aggatagaat
     2641 cagcaaaaaa aagaaagagt atctgctgga agaacgggac atcaacaagt tcgaggtgca
     2701 aaaagagttc atcaatagaa acctggtgga tacccggtac gccacaagag agctgacaaa
     2761 ctacctgaag gcctacttca gcgccaacaa tatgaacgtg aaggtgaaaa cgatcaacgg
     2821 cagcttcacc gattacctgc ggaaagtgtg gaagtttaag aaggaacgga accacggcta
     2881 caagcaccac gccgaggacg ccctgattat cgctaatgcc gatttcctgt tcaaagagaa
     2941 caagaagctg aaagccgtga actctgtgct ggaaaaacct gagatcgaga gcaagcagct
     3001 ggatatccag gtggatagcg aggataacta cagcgaaatg ttcatcatcc ctaagcaggt
     3061 ccaggacatc aaggacttca gaaacttcaa gtacagccac agagtggaca agaagcctaa
     3121 cagacagctg atcaacgata cactgtacag cacccggaag aaggacaact ccacctacat
     3181 cgtgcagacc atcaaagata tctatgccaa agataatacc accctgaaga agcagtttga
     3241 caagtcaccc gagaagttcc tcatgtacca acacgatccg cggaccttcg agaagttgga
     3301 agtgatcatg aagcagtacg ctaatgagaa gaatcctctg gccaagtacc acgaggaaac
     3361 aggcgagtac ctgaccaaat acagcaaaaa aaacaacggc cctatcgtga aaagcctgaa
     3421 gtacattgga aacaagctgg gcagccacct agatgtgacc caccagttca agagcagcac
     3481 caagaagttg gtgaagctga gcatcaagcc ttatagattc gacgtctacc tgaccgacaa
     3541 gggatataag ttcatcacca tcagctacct ggacgtgctg aagaaagaca attactacta
     3601 catacccgaa cagaagtacg acaagctcaa actgggcaag gccatcgaca aaaacgccaa
     3661 gtttatcgct agcttctaca agaatgatct gatcaagctg gacggcgaga tctacaagat
     3721 catcggcgtg aatagcgaca ccagaaacat gatcgaactg gatctgcctg acatcagata
     3781 caaagaatac tgcgagctga acaatatcaa gggcgaacct agaatcaaaa agaccatcgg
     3841 caaaaaggtg aatagcatcg aaaaactgac aaccgacgtg ctgggcaacg tgttcaccaa
     3901 cacccagtac acaaaacctc agctgctgtt caagcgagga aataaaaggc cggcggccac
     3961 gaaaaaggcc ggccaggcaa aaaagaaaaa gggatcctac ccatacgatg ttccagatta
     4021 cgcttaccca tacgatgttc cagattacgc ttacccatac gatgttccag attacgctta
     4081 agaattccta gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     4141 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     4201 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     4261 ggggtggggc aggacagcaa gggggaggat tgggaagaga atagcaggca tgctggggag
     4321 gtaccgaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta
     4381 gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac
     4441 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact
     4501 atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga
     4561 aaggacgaaa caccggagac cacggcaggt ctcagtttta gtactctgga aacagaatct
     4621 actaaaacaa ggcaaaatgc cgtgtttatc tcgtcaactt gttggcgaga tttttgcggc
     4681 cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg
     4741 aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg
     4801 agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt
     4861 gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt
     4921 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc
     4981 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     5041 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     5101 caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     5161 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     5221 aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt
     5281 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     5341 aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     5401 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     5461 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     5521 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     5581 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     5641 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     5701 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     5761 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     5821 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     5881 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     5941 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     6001 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     6061 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     6121 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     6181 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     6241 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     6301 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     6361 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     6421 gtttattgct gataaatctg gagccggtga gcgtggaagc cgcggtatca ttgcagcact
     6481 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     6541 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     6601 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     6661 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     6721 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     6781 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     6841 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     6901 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     6961 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     7021 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     7081 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     7141 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     7201 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     7261 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     7321 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     7381 tttacggttc ctggcctttt gctggccttt tgctcacatg tcctgcaggc agctgcgcgc
     7441 tcgctcgct
//
