LOCUS       sequence-328007-        7449 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 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-HF
FEATURES             Location/Qualifiers
     source          1..7449
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      223..232
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             235..255
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3439..3486
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3493..3573
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(3601..3618)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3607..3814
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3821..4069
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3821..3841
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3992..4011
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4090..4165
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4179..4319
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4179..4308
                     /label=AAV2 ITR
     rep_origin      4394..4849
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4481..4500)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4691..4712
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4866..4885)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4985..5007
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5045..5063)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5131..5235
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5236..5304,5305..6096)
                     /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(5454..5473)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6267..6855
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6756..6775
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   6917..7046
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        7070..7449
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctctctgg ctaactaccg gtgccaccat ggccccaaag
      241 aagaagcgga aggtcggtat ccacggagtc ccagcagcca accaaaaatt catactggga
      301 ctggacatcg gaatcaccag cgtgggctac ggcctgatcg actacgagac aaagaatatc
      361 atcgatgccg gcgttagact gttccccgag gccaacgtgg aaaacaacga gggaagaagg
      421 tccaaacgtg gaagcagaag actgaagcga cgccgcattc acagacttga acgggtgaag
      481 aagctgctcg aggattataa tctgctggat cagtcccaga ttcctcagtc tacaaacccc
      541 tacgccatcc gcgtgaaggg cctgtctgaa gccctgagca aggacgaact cgtgattgcc
      601 ctgctccata tcgccaagag aagaggcatc cacaagatcg acgtgatcga cagcaacgac
      661 gacgtgggga acgagctcag caccaaggaa cagctgaata agaacagcaa gctgctgaaa
      721 gacaaatttg tgtgccagat ccagctggaa agaatgaatg agggccaggt gcggggagag
      781 aaaaaccggt tcaagaccgc tgatatcatc aaggaaatca tccagctgct gaatgtgcag
      841 aagaacttcc accagctgga cgagaacttc atcaacaagt acatcgaact ggttgagatg
      901 agacgggaat acttcgaggg ccccggcaag ggcagtccat atggctggga aggcgaccct
      961 aaggcttggt acgagacact gatgggccac tgcacctact tcccagatga gctggctagc
     1021 gtgaaatacg cctacagtgc cgacctgttc aacgctctga acgacctgaa caacctggtc
     1081 atccaaagag atggactgtc taagctcgag tatcatgaga agtatcacat catcgagaac
     1141 gtgttcaagc agaagaagaa acctacactg aagcagatcg ccaatgagat caatgtcaac
     1201 cctgaagata tcaagggcta cagaatcaca aagtctggca agccccagtt taccgagttt
     1261 aagctctacc acgacctgaa aagcgtgctg tttgaccaga gcatcctgga gaacgaagac
     1321 gtgctggacc agatcgctga gatcctgacc atctaccagg acaaggatag catcaaatct
     1381 aagctgacgg aactggacat cctgctgaac gaggaagata aggaaaacat cgcccagctg
     1441 actggctaca ccgggaccca ccggctcagc ctgaaatgca tccggctggt cctggaagag
     1501 cagtggtatt ctagccgggc tcagatggaa atcttcgccc acctgaacat taagcctaag
     1561 aagatcaacc tgacagccgc caacaagatc ccgaaggcta tgatcgacga gttcatcctg
     1621 agccctgtgg tgaagaggac cttcggccag gccattaacc ttattaacaa gatcatagaa
     1681 aagtacggcg tgcctgaaga tatcatcatc gagctggcca gagaaaataa tagcaaggac
     1741 aagcagaagt tcatcaatga gatgcagaaa aagaacgaga acaccagaaa gagaattaac
     1801 gaaatcatcg gcaagtatgg caaccagaac gccaagagac tggtcgagaa gattagactg
     1861 cacgacgagc aggagggcaa gtgcctgtac tcactggaaa gcatccctct ggaggacctg
     1921 ctgaacaacc ccaaccacta cgaggtggac cacatcattc caagatctgt gtccttcgac
     1981 aactcttacc acaacaaagt gctcgtgaag cagagcgaga actccaaaaa atccaacctg
     2041 accccttacc agtactttaa cagcggcaag tccaagctct cttacaacca gtttaaacaa
     2101 cacatcctga acctgagcaa gtcccaggat agaatcagca aaaaaaagaa agagtatctg
     2161 ctggaagaac gggacatcaa caagttcgag gtgcaaaaag agttcatcaa tagaaacctg
     2221 gtggataccc ggtacgccac agccgagctg acaaactacc tgaaggccta cttcagcgcc
     2281 aacaatatga acgtgaaggt gaaaacgatc aacggcagct tcaccgatta cctgcggaaa
     2341 gtgtggaagt ttaagaagga acggaaccac ggctacaagc accacgccga ggacgccctg
     2401 attatcgcta atgccgattt cctgttcaaa gagaacaaga agctgaaagc cgtgaactct
     2461 gtgctggaaa aacctgagat cgagagcaag cagctggata tccaggtgga tagcgaggat
     2521 aactacagcg aaatgttcat catccctaag caggtccagg acatcaagga cttcagaaac
     2581 ttcaagtaca gccacagagt ggacaagaag cctaacagac agctgatcaa cgatacactg
     2641 tacagcaccc ggaagaagga caactccacc tacatcgtgc agaccatcaa agatatctat
     2701 gccaaagata ataccaccct gaagaagcag tttgacaagt cacccgagaa gttcctcatg
     2761 taccaacacg atccgcggac cttcgagaag ttggaagtga tcatgaagca gtacgctaat
     2821 gagaagaatc ctctggccaa gtaccacgag gaaacaggcg agtacctgac caaatacagc
     2881 aaaaaaaaca acggccctat cgtgaaaagc ctgaagtaca ttggaaacaa gctgggcagc
     2941 cacctagatg tgacccacca gttcaagagc agcaccaaga agttggtgaa gctgagcatc
     3001 aagccttata gattcgacgt ctacctgacc gacaagggat ataagttcat caccatcagc
     3061 tacctggacg tgctgaagaa agacaattac tactacatac ccgaacagaa gtacgacaag
     3121 ctcaaactgg gcaaggccat cgacaaaaac gccaagttta tcgctagctt ctacaagaat
     3181 gatctgatca agctggacgg cgagatctac aagatcatcg gcgtgaatag cgacaccaga
     3241 aacatgatcg aactggatct gcctgacatc agatacaaag aatactgcga gctgaacaat
     3301 atcaagggcg aacctagaat caaaaagacc atcggcaaaa aggtgaatag catcgaaaaa
     3361 ctgacaaccg acgtgctggg caacgtgttc accaacaccc agtacacaaa acctcagctg
     3421 ctgttcaagc gaggaaataa aaggccggcg gccacgaaaa aggccggcca ggcaaaaaag
     3481 aaaaagggat cctacccata cgatgttcca gattacgctt acccatacga tgttccagat
     3541 tacgcttacc catacgatgt tccagattac gcttaagaat tcctagagct cgctgatcag
     3601 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     3661 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     3721 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     3781 aggattggga agagaatagc aggcatgctg gggaggtacc gagggcctat ttcccatgat
     3841 tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac
     3901 tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta
     3961 gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa
     4021 gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg gagaccacgg
     4081 caggtctcag ttttagtact ctggaaacag aatctactaa aacaaggcaa aatgccgtgt
     4141 ttatctcgtc aacttgttgg cgagattttt gcggccgcag gaacccctag tgatggagtt
     4201 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg
     4261 acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg
     4321 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca
     4381 aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     4441 cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct
     4501 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     4561 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt
     4621 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     4681 ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat
     4741 tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt
     4801 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact
     4861 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     4921 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     4981 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     5041 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     5101 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     5161 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     5221 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     5281 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     5341 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     5401 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     5461 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     5521 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     5581 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     5641 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     5701 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     5761 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     5821 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     5881 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     5941 ggtgagcgtg gaagccgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     6001 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     6061 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     6121 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     6181 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     6241 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     6301 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     6361 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     6421 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     6481 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     6541 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     6601 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     6661 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     6721 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     6781 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     6841 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     6901 ccttttgctc acatgtcctg caggcagctg cgcgctcgct cgctcactga ggccgcccgg
     6961 gcgtcgggcg acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt
     7021 ggccaactcc atcactaggg gttcctgcgg cctctagact cgaggcgttg acattgatta
     7081 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
     7141 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
     7201 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
     7261 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
     7321 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
     7381 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
     7441 attaccatg
//
