LOCUS       sequence-339405-        8669 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses nSaCas9 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8669)
  AUTHORS   Wang L, Xue W, Zhang H, Gao R, Qiu H, Wei J, Zhou L, Lei YN, Wu X, 
            Li X, Liu C, Wu J, Chen Q, Ma H, Huang X, Cai C, Zhang Y, Yang B, 
            Yin H, Yang L, Chen J
  TITLE     Eliminating base-editor-induced genome-wide and transcriptome-wide 
            off-target mutations.
  JOURNAL   Nat Cell Biol. 2021 May 10. pii: 10.1038/s41556-021-00671-4. doi: 
            10.1038/s41556-021-00671-4.
  PUBMED    33972728
REFERENCE   2  (bases 1 to 8669)
  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: "Nat Cell 
            Biol. 2021 May 10. pii: 10.1038/s41556-021-00671-4. doi: 
            10.1038/s41556-021-00671-4."
COMMENT     Sequence Label: pU6-SasgRNA scaffold-CMV-nSaCas9
FEATURES             Location/Qualifiers
     source          1..8669
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        235..483
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     235..255
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     406..425
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        633..1012
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1013..1216
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1167..1187
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1261..1280
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1261..1279
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1297..1306
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1306..1329
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4498..4518
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4534..4575
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             4585..4602
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4625..4642)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4631..4855
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4901..5329
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4988..5007)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5198..5219
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5338..5358)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5343..5672
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5523..5658
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5585..5604
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        5720..5767
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5786..6184
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    6342..6463
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6379..6398)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6433..6452
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6512..6528)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6512..6528)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6525..6547)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6536..6552
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(6560..6566,6567..6584,6585..6590))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6605..6626
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6743..6760)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6914..7502)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6994..7013)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7673..8464,8465..8533))
                     /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     8296..8315
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8534..8638)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgagggc
      241 ctatttccca tgattccttc atatttgcat atacgataca aggctgttag agagataatt
      301 ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa
      361 taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt
      421 accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac
      481 accgggagac ggagagagcg tctccgtttt agtactctgt aattttaggt atgaggtaga
      541 cgaaaattgt acttatacct aaaattacag aatctactaa aacaaggcaa aatgccgtgt
      601 ttatctcgtc aacttgttgg cgagattttt ttgacattga ttattgacta gttattaata
      661 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
      721 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
      781 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
      841 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
      901 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg
      961 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg
     1021 gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct
     1081 ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa
     1141 atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt
     1201 ctatataagc agagctctct ggctaactag agaacccact gcttactggc ttatcgaaat
     1261 taatacgact cactataggg agacccaagc tggctagcca ccatggacta caaagatgat
     1321 gacgacaaag gcagcaagcg gaactatatc ctcgggctgg ctattggcat cacatctgtc
     1381 ggctatggta taatagacta tgaaacaagg gacgtgattg acgcaggtgt gaggctgttc
     1441 aaggaggcaa acgtcgagaa caacgaaggt cggagaagca agaggggtgc ccggaggctg
     1501 aagaggagga gaaggcacag aatacagcgg gtcaagaaac tcctgttcga ctataacctg
     1561 ctgaccgatc attccgaact gtcaggcatc aatccttacg aagccagagt caagggtctg
     1621 tctcaaaaac tctctgagga agagttttcc gcagccctgc tgcacctggc taagaggaga
     1681 ggagtccaca acgtcaatga ggttgaggag gatacaggga acgaactgtc tacaaaggaa
     1741 cagatcagcc ggaatagcaa ggccctggaa gagaagtacg ttgctgaact gcagctggaa
     1801 aggctcaaga aagatggaga ggttcggggt tccatcaaca ggttcaagac atctgactat
     1861 gtgaaggaag ccaagcaact gctcaaggtg cagaaggcct accatcagct cgaccagagc
     1921 ttcattgata cttacataga cctgctggag actaggagaa cttactacga agggcctggc
     1981 gagggcagcc ctttcggctg gaaagatatc aaggagtggt acgagatgct catggggcat
     2041 tgcacctact tccccgaaga actgaggtca gtcaagtacg cctacaacgc agacctgtac
     2101 aacgccctga atgatctcaa caatctcgtc ataactcggg atgaaaacga gaagctggaa
     2161 tattatgaga agttccagat tattgaaaat gtgttcaaac agaagaagaa acctaccctg
     2221 aaacaaattg ccaaagagat cctggtgaat gaggaggata tcaagggata tcgggttact
     2281 tctaccggca aaccagagtt cacaaatctg aaagtttacc atgacatcaa agatattacc
     2341 gcaagaaagg agatcatcga gaacgctgag ctcctggacc agatcgctaa gattctcact
     2401 atctatcagt ccagcgagga tattcaggaa gagctgacca acctgaactc agagctgact
     2461 caggaagaaa tcgaacaaat ctccaatctg aaaggataca ctggtaccca taatctctca
     2521 ctcaaggcta tcaatctgat cctggatgaa ctgtggcata ctaacgacaa tcagatcgcc
     2581 atcttcaatc ggctcaaact ggtgcccaaa aaagtggacc tgagccaaca gaaagagatt
     2641 cctacaaccc tggtggacga tttcattctg agcccagtgg ttaagcggag cttcatccaa
     2701 tccatcaagg tgatcaacgc tatcatcaag aagtatggcc tgcctaatga cataatcatt
     2761 gaactcgcaa gggaaaagaa tagcaaagat gcccagaaga tgataaacga gatgcagaaa
     2821 cggaacagac agactaacga aagaatcgag gaaataatac ggaccactgg taaggagaac
     2881 gctaagtatc tgatcgagaa aatcaagctg cacgatatgc aggaaggcaa gtgcctgtat
     2941 tctctggagg ctatacccct ggaggatctg ctcaataatc ctttcaatta cgaggtggat
     3001 cacatcatac caagatccgt gagctttgac aatagcttta ataataaggt gctcgtgaag
     3061 caggaggaaa actcaaagaa aggcaacagg accccattcc agtacctgtc cagctctgac
     3121 agcaagatta gctacgaaac cttcaagaaa cacatcctga atctggccaa gggcaaggga
     3181 agaataagca aaacaaagaa agagtatctc ctggaggaaa gggacatcaa caggttttca
     3241 gtgcagaaag attttatcaa tcggaatctc gttgacacaa gatatgctac cagagggctc
     3301 atgaatctgc tcaggtcata ctttagggtg aacaacctgg atgtgaaggt caaatccata
     3361 aatggagggt tcacttcctt tctcaggaga aaatggaagt ttaagaaaga gagaaataag
     3421 ggttacaaac atcacgccga ggacgcactg atcattgcca acgctgactt tatctttaag
     3481 gaatggaaga agctggacaa agcaaagaag gtgatggaga atcagatgtt tgaggaaaag
     3541 caggccgagt ctatgcctga gattgaaaca gagcaggaat acaaagagat cttcattact
     3601 ccacatcaga ttaagcacat caaggacttt aaggactata aatactcaca tagggtggat
     3661 aagaaaccta atagagagct gatcaacgat acactctact caacaaggaa agacgacaaa
     3721 ggaaacaccc tgattgttaa taatctcaat gggctgtatg acaaagataa tgacaagctg
     3781 aagaaactca tcaacaagtc cccagaaaag ctgctgatgt atcaccacga tccccaaaca
     3841 tatcagaagc tgaagctgat tatggagcag tatggtgatg agaagaaccc tctgtacaag
     3901 tactatgaag agacagggaa ctacctcact aagtacagca agaaagacaa cggacccgtt
     3961 atcaagaaga tcaagtacta cggcaataag ctgaacgccc acctggatat cacagatgac
     4021 tatccaaact ctaggaacaa agtggtgaaa ctgtccctga agccatacag atttgatgtg
     4081 tatctggata acggagtcta taagttcgtc acagtcaaga acctggacgt catcaagaag
     4141 gagaattact atgaagtgaa cagcaaatgc tacgaggaag ccaagaagct caagaagatt
     4201 tctaaccagg cagagtttat cgcctctttc tacaataacg atctgatcaa gatcaacgga
     4261 gaactgtaca gagtgatcgg cgtgaataat gacctcctga ataggatcga ggttaacatg
     4321 atcgatatca catatcggga gtacctggag aatatgaatg acaagaggcc tcccagaatt
     4381 atcaagacta ttgccagcaa aacccaatct ataaaaaagt actcaacaga tatcctgggg
     4441 aacctgtatg aggtgaagtc aaagaagcat ccccagatta tcaagaaagg cggaagccca
     4501 aagaagaaac ggaaagtcta agggcccttc gaaggtaagc ctatccctaa ccctctcctc
     4561 ggtctcgatt ctacgcgtac cggtcatcat caccatcacc attgagttta aacccgctga
     4621 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     4681 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     4741 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     4801 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
     4861 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
     4921 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     4981 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     5041 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     5101 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc aatcgccctg atagacggtt
     5161 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     5221 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     5281 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga
     5341 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     5401 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     5461 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     5521 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     5581 tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag
     5641 gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt
     5701 tcggatctga tcagcacgtg ttgacaatta atcatcggca tagtatatcg gcatagtata
     5761 atacgacaag gtgaggaact aaaccatggc caagcctttg tctcaagaag aatccaccct
     5821 cattgaaaga gcaacggcta caatcaacag catccccatc tctgaagact acagcgtcgc
     5881 cagcgcagct ctctctagcg acggccgcat cttcactggt gtcaatgtat atcattttac
     5941 tgggggacct tgtgcagaac tcgtggtgct gggcactgct gctgctgcgg cagctggcaa
     6001 cctgacttgt atcgtcgcga tcggaaatga gaacaggggc atcttgagcc cctgcggacg
     6061 gtgccgacag gtgcttctcg atctgcatcc tgggatcaaa gccatagtga aggacagtga
     6121 tggacagccg acggcagttg ggattcgtga attgctgccc tctggttatg tgtgggaggg
     6181 ctaagcactt cgtggccgag gagcaggact gacacgtgct acgagatttc gattccaccg
     6241 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     6301 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt
     6361 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     6421 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
     6481 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     6541 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     6601 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     6661 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     6721 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     6781 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     6841 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     6901 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     6961 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     7021 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     7081 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     7141 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     7201 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     7261 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     7321 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     7381 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     7441 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     7501 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     7561 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     7621 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     7681 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     7741 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     7801 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     7861 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     7921 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     7981 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     8041 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     8101 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     8161 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     8221 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     8281 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     8341 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     8401 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     8461 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     8521 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     8581 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     8641 catttccccg aaaagtgcca cctgacgtc
//
