LOCUS       sequence-328013-        7452 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Sa-SlugCas9, and cloning backbone for sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7452)
  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 7452)
  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: Sa-SlugCas9
FEATURES             Location/Qualifiers
     source          1..7452
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        105..308
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     259..279
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      327..336
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             339..359
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3546..3593
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3600..3680
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(3708..3725)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3714..3921
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3928..4176
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3928..3948
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4099..4118
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4197..4272
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4286..4426
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4286..4415
                     /label=AAV2 ITR
     rep_origin      4501..4956
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4588..4607)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4798..4819
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4973..4992)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5092..5114
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5152..5170)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5238..5342
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5343..5411,5412..6203)
                     /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(5561..5580)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6374..6962
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6863..6882
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   7024..7153
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        join(7177..7452,1..104)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
       61 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
      121 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
      181 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
      241 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
      301 gcagagctct ctggctaact accggtgcca ccatggcccc aaagaagaag cggaaggtcg
      361 gtatccacgg agtcccagca gccaagcgga actacatcct gggcctggac atcggcatca
      421 ccagcgtggg ctacggcatc atcgactacg agacacggga cgtgatcgat gccggcgtgc
      481 ggctgttcaa agaggccaac gtggaaaaca acgagggcag gcggagcaag agaggcgcca
      541 gaaggctgaa gcggcggagg cggcatagaa tccagagagt gaagaagctg ctgttcgact
      601 acaacctgct gaccgaccac agcgagctga gcggcatcaa cccctacgag gccagagtga
      661 agggcctgag ccagaagctg agcgaggaag agttctctgc cgccctgctg cacctggcca
      721 agagaagagg cgtgcacaac gtgaacgagg tggaagagga caccggcaac gagctgtcca
      781 ccaaagagca gatcagccgg aacagcaagg ccctggaaga gaaatacgtg gccgaactgc
      841 agctggaacg gctgaagaaa gacggcgaag tgcggggcag catcaacaga ttcaagacca
      901 gcgactacgt gaaagaagcc aaacagctgc tgaaggtgca gaaggcctac caccagctgg
      961 accagagctt catcgacacc tacatcgacc tgctggaaac ccggcggacc tactatgagg
     1021 gacctggcga gggcagcccc ttcggctgga aggacatcaa agaatggtac gagatgctga
     1081 tgggccactg cacctacttc cccgaggaac tgcggagcgt gaagtacgcc tacaacgccg
     1141 acctgtacaa cgccctgaac gacctgaaca atctcgtgat caccagggac gagaacgaga
     1201 agctggaata ttacgagaag ttccagatca tcgagaacgt gttcaagcag aagaagaagc
     1261 ccaccctgaa gcagatcgcc aaagaaatcc tcgtgaacga agaggatatt aagggctaca
     1321 gagtgaccag caccggcaag cccgagttca ccaacctgaa ggtgtaccac gacatcaagg
     1381 acattaccgc ccggaaagag attattgaga acgccgagct gctggatcag attgccaaga
     1441 tcctgaccat ctaccagagc agcgaggaca tccaggaaga actgaccaat ctgaactccg
     1501 agctgaccca ggaagagatc gagcagatct ctaatctgaa gggctatacc ggcacccaca
     1561 acctgagcct gaaggccatc aacctgatcc tggacgagct gtggcacacc aacgacaacc
     1621 agatcgctat cttcaaccgg ctgaagctgg tgcccaagaa ggtggacctg tcccagcaga
     1681 aagagatccc caccaccctg gtggacgact tcatcctgag ccccgtcgtg aagagaagct
     1741 tcatccagag catcaaagtg atcaacgcca tcatcaagaa gtacggcctg cccaacgaca
     1801 tcattatcga gctggcccgc gagaagaact ccaaggacgc ccagaaaatg atcaacgaga
     1861 tgcagaagcg gaaccggcag accaacgagc ggatcgagga aatcatccgg accaccggca
     1921 aagagaacgc caagtacctg atcgagaaga tcaagctgca cgacatgcag gaaggcaagt
     1981 gcctgtacag cctggaagcc atccctctgg aagatctgct gaacaacccc ttcaactatg
     2041 aggtggacca catcatcccc agaagcgtgt ccttcgacaa cagcttcaac aacaaggtgc
     2101 tcgtgaagca ggaagaaaac agcaagaagg gcaaccggac cccattccag tacctgagca
     2161 gcagcgacag caagatcagc tacgaaacct tcaagaagca catcctgaat ctggccaagg
     2221 gcaagggcag aatcagcaag accaagaaag agtatctgct ggaagaacgg gacatcaaca
     2281 ggttctccgt gcagaaagac ttcatcaacc ggaacctggt ggataccaga tacgccacca
     2341 gaggcctgat gaacctgctg cggagctact tcagagtgaa caacctggac gtgaaagtga
     2401 agtccatcaa tggcggcttc accagctttc tgcggcggaa gtggaagttt aagaaagagc
     2461 ggaacaaggg gtacaagcac cacgccgagg acgccctgat cattgccaac gccgatttca
     2521 tcttcaaaga gtggaagaaa ctggacaagg ccaaaaaagt gatggaaaac cagatgttcg
     2581 aggaaaagca ggccgagagc atgcccgaga tcgaaaccga gcaggagtac aaagagatct
     2641 tcatcacccc ccaccagatc aagcacatta aggacttcaa ggactacaag tacagccacc
     2701 gggtggacaa gaagcctaat agagagctga ttaacgacac cctgtactcc acccggaagg
     2761 acgacaaggg caacaccctg atcgtgaaca atctgaacgg cctgtacgac aaggacaatg
     2821 acaagctgaa aaagctgatc aacaagagcc ccgaaaagct gctgatgtac caccacgacc
     2881 cccagaccta ccagaaactg aagctgatta tggaacagta cggcgacgag aagaatcccc
     2941 tgtacaagta ctacgaggaa accgggaact acctgaccaa gtactccaaa aaggacaacg
     3001 gccccgtgat caagaagatt aagtattacg gcaacaaact gaacgcccat ctggacatca
     3061 ccgacgacta ccccaacagc agaaacaagg tcgtgaagct gtccctgaag ccctaccgct
     3121 tcgacgtgta cctgaccgac aagggctaca agttcatcac catcagctac ctggacgtgc
     3181 tgaagaagga caactactac tacatccccg agcagaagta cgacaagctg aagctgggca
     3241 aggccatcga caagaacgcc aagttcatcg ccagcttcta caagaacgac ctgataaagc
     3301 tggacggcga gatatacaag ataatcggcg tgaacagcga cacccgcaac atgatagagc
     3361 tggacctgcc cgacatccgc tacaaggagt actgcgagct gaacaacatc aagggcgagc
     3421 cccgcatcaa gaagaccatc ggcaagaagg tgaacagcat cgagaagctg accaccgacg
     3481 tgctgggcaa cgtgttcacc aacacccagt acaccaagcc ccagctgctg ttcaagcgcg
     3541 gcaacaaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa aagggatcct
     3601 acccatacga tgttccagat tacgcttacc catacgatgt tccagattac gcttacccat
     3661 acgatgttcc agattacgct taagaattcc tagagctcgc tgatcagcct cgactgtgcc
     3721 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     3781 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     3841 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     3901 gaatagcagg catgctgggg aggtaccgag ggcctatttc ccatgattcc ttcatatttg
     3961 catatacgat acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag
     4021 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
     4081 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
     4141 ttggctttat atatcttgtg gaaaggacga aacaccggag accacggcag gtctcagttt
     4201 tagtactctg gaaacagaat ctactaaaac aaggcaaaat gccgtgttta tctcgtcaac
     4261 ttgttggcga gatttttgcg gccgcaggaa cccctagtga tggagttggc cactccctct
     4321 ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt
     4381 gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg
     4441 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt
     4501 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     4561 ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     4621 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     4681 gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc
     4741 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     4801 gactcttgtt ccaaactgga acaacactca actctatctc gggctattct tttgatttat
     4861 aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta
     4921 acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct
     4981 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     5041 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     5101 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     5161 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     5221 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     5281 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     5341 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     5401 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     5461 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     5521 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     5581 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     5641 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     5701 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     5761 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     5821 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     5881 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     5941 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     6001 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggaa
     6061 gccgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     6121 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     6181 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     6241 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     6301 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     6361 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     6421 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     6481 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     6541 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     6601 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     6661 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     6721 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     6781 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     6841 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     6901 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     6961 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     7021 tgtcctgcag gcagctgcgc gctcgctcgc tcactgaggc cgcccgggcg tcgggcgacc
     7081 tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc
     7141 actaggggtt cctgcggcct ctagactcga ggcgttgaca ttgattattg actagttatt
     7201 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
     7261 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
     7321 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
     7381 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
     7441 cccctattga cg
//
