LOCUS       sequence-358845-        6930 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express SaCas9-mSA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6930)
  AUTHORS   Liang SQ, Liu P, Smith JL, Mintzer E, Maitland S, Dong X, Yang Q, 
            Lee J, Haynes CM, Zhu LJ, Watts JK, Sontheimer EJ, Wolfe SA, Xue W
  TITLE     Genome-wide detection of CRISPR editing in vivo using GUIDE-tag.
  JOURNAL   Nat Commun. 2022 Jan 21;13(1):437. doi: 10.1038/s41467-022-28135-9.
  PUBMED    35064134
REFERENCE   2  (bases 1 to 6930)
  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 
            Commun."; date: "2022-01-21"; volume: "13(1):437. doi"; pages: " 
            10.1038/s41467-022-28135-9"
COMMENT     Sequence Label: SaCas9-mSA
FEATURES             Location/Qualifiers
     source          1..6930
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..721
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     672..692
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          813..945
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     975..994
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        975..993
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1014..1023
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1029..1055
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             1092..1118
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             1185..4340
                     /codon_start=1
                     /product="Cas9 endonuclease from the Staphylococcus aureus 
                     Type II CRISPR/Cas system"
                     /label=SaCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG
                     RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF
                     SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV
                     RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW
                     KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ
                     IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI
                     IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
                     NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK
                     VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK
                     YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ
                     EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS
                     VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN
                     KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF
                     ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN
                     DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD
                     NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL
                     DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN
                     RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI
                     IKKG"
     CDS             4353..4373
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4374..4463
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             4476..4502
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     CDS             4545..4886
                     /codon_start=1
                     /product="monomeric streptavidin/rhizavidin hybrid (Lim et 
                     al., 2013)"
                     /label=mSA
                     /translation="AEAGITGTWYNQSGSTFTVTAGADGNLTGQYENRAQGTGCQNSPY
                     TLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATE
                     QGQDTFTKVK"
     primer_bind     complement(4915..4931)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4915..4931)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             4944..4970
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             5013..5039
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     primer_bind     complement(5103..5122)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    5132..5213
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     5157..5176
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(5411..5999)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5491..5510)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6080..6874)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(6080..6874)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6191..6210)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6801..6820
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      661 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagg
      721 tcgtttagtg aaccgtcaga tcactagtag ctttattgcg gtagtttatc acagttaaat
      781 tgctaacgca gtcagtgctc gactgatcac aggtaagtat caaggttaca agacaggttt
      841 aaggaggcca atagaaactg ggcttgtcga gacagagaag attcttgcgt ttctgatagg
      901 cacctattgg tcttactgac atccactttg cctttctctc cacaggggta ccgaagccgc
      961 tagcgctacc ggtctaatac gactcactat agggcgaatt gggtaccggg cccgccacca
     1021 tggctagccc cgccgccaag cgcgtgaagc tggacggcgg cagcggcggc ggcagcggcg
     1081 gcggcagcgg cccggcagct aagagggtga aactggatgg cggtagcggc ggaggcagcg
     1141 gtggcggcag cggccccctc gagatccacg gagtcccagc agccaagcgg aactacatcc
     1201 tgggcctgga catcggcatc accagcgtgg gctacggcat catcgactac gagacacggg
     1261 acgtgatcga tgccggcgtg cggctgttca aagaggccaa cgtggaaaac aacgagggca
     1321 ggcggagcaa gagaggcgcc agaaggctga agcggcggag gcggcataga atccagagag
     1381 tgaagaagct gctgttcgac tacaacctgc tgaccgacca cagcgagctg agcggcatca
     1441 acccctacga ggccagagtg aagggcctga gccagaagct gagcgaggaa gagttctctg
     1501 ccgccctgct gcacctggcc aagagaagag gcgtgcacaa cgtgaacgag gtggaagagg
     1561 acaccggcaa cgagctgtcc accaaagagc agatcagccg gaacagcaag gccctggaag
     1621 agaaatacgt ggccgaactg cagctggaac ggctgaagaa agacggcgaa gtgcggggca
     1681 gcatcaacag attcaagacc agcgactacg tgaaagaagc caaacagctg ctgaaggtgc
     1741 agaaggccta ccaccagctg gaccagagct tcatcgacac ctacatcgac ctgctggaaa
     1801 cccggcggac ctactatgag ggacctggcg agggcagccc cttcggctgg aaggacatca
     1861 aagaatggta cgagatgctg atgggccact gcacctactt ccccgaggaa ctgcggagcg
     1921 tgaagtacgc ctacaacgcc gacctgtaca acgccctgaa cgacctgaac aatctcgtga
     1981 tcaccaggga cgagaacgag aagctggaat attacgagaa gttccagatc atcgagaacg
     2041 tgttcaagca gaagaagaag cccaccctga agcagatcgc caaagaaatc ctcgtgaacg
     2101 aagaggatat taagggctac agagtgacca gcaccggcaa gcccgagttc accaacctga
     2161 aggtgtacca cgacatcaag gacattaccg cccggaaaga gattattgag aacgccgagc
     2221 tgctggatca gattgccaag atcctgacca tctaccagag cagcgaggac atccaggaag
     2281 aactgaccaa tctgaactcc gagctgaccc aggaagagat cgagcagatc tctaatctga
     2341 agggctatac cggcacccac aacctgagcc tgaaggccat caacctgatc ctggacgagc
     2401 tgtggcacac caacgacaac cagatcgcta tcttcaaccg gctgaagctg gtgcccaaga
     2461 aggtggacct gtcccagcag aaagagatcc ccaccaccct ggtggacgac ttcatcctga
     2521 gccccgtcgt gaagagaagc ttcatccaga gcatcaaagt gatcaacgcc atcatcaaga
     2581 agtacggcct gcccaacgac atcattatcg agctggcccg cgagaagaac tccaaggacg
     2641 cccagaaaat gatcaacgag atgcagaagc ggaaccggca gaccaacgag cggatcgagg
     2701 aaatcatccg gaccaccggc aaagagaacg ccaagtacct gatcgagaag atcaagctgc
     2761 acgacatgca ggaaggcaag tgcctgtaca gcctggaagc catccctctg gaagatctgc
     2821 tgaacaaccc cttcaactat gaggtggacc acatcatccc cagaagcgtg tccttcgaca
     2881 acagcttcaa caacaaggtg ctcgtgaagc aggaagaaaa cagcaagaag ggcaaccgga
     2941 ccccattcca gtacctgagc agcagcgaca gcaagatcag ctacgaaacc ttcaagaagc
     3001 acatcctgaa tctggccaag ggcaagggca gaatcagcaa gaccaagaaa gagtatctgc
     3061 tggaagaacg ggacatcaac aggttctccg tgcagaaaga cttcatcaac cggaacctgg
     3121 tggataccag atacgccacc agaggcctga tgaacctgct gcggagctac ttcagagtga
     3181 acaacctgga cgtgaaagtg aagtccatca atggcggctt caccagcttt ctgcggcgga
     3241 agtggaagtt taagaaagag cggaacaagg ggtacaagca ccacgccgag gacgccctga
     3301 tcattgccaa cgccgatttc atcttcaaag agtggaagaa actggacaag gccaaaaaag
     3361 tgatggaaaa ccagatgttc gaggaaaagc aggccgagag catgcccgag atcgaaaccg
     3421 agcaggagta caaagagatc ttcatcaccc cccaccagat caagcacatt aaggacttca
     3481 aggactacaa gtacagccac cgggtggaca agaagcctaa tagagagctg attaacgaca
     3541 ccctgtactc cacccggaag gacgacaagg gcaacaccct gatcgtgaac aatctgaacg
     3601 gcctgtacga caaggacaat gacaagctga aaaagctgat caacaagagc cccgaaaagc
     3661 tgctgatgta ccaccacgac ccccagacct accagaaact gaagctgatt atggaacagt
     3721 acggcgacga gaagaatccc ctgtacaagt actacgagga aaccgggaac tacctgacca
     3781 agtactccaa aaaggacaac ggccccgtga tcaagaagat taagtattac ggcaacaaac
     3841 tgaacgccca tctggacatc accgacgact accccaacag cagaaacaag gtcgtgaagc
     3901 tgtccctgaa gccctacaga ttcgacgtgt acctggacaa tggcgtgtac aagttcgtga
     3961 ccgtgaagaa tctggatgtg atcaaaaaag aaaactacta cgaagtgaat agcaagtgct
     4021 atgaggaagc taagaagctg aagaagatca gcaaccaggc cgagtttatc gcctccttct
     4081 acaacaacga tctgatcaag atcaacggcg agctgtatag agtgatcggc gtgaacaacg
     4141 acctgctgaa ccggatcgaa gtgaacatga tcgacatcac ctaccgcgag tacctggaaa
     4201 acatgaacga caagaggccc cccaggatca ttaagacaat cgcctccaag acccagagca
     4261 ttaagaagta cagcacagac attctgggca acctgtatga agtgaaatct aagaagcacc
     4321 ctcagatcat caaaaagggc ggcaccggcg ggcccaagaa gaagaggaag gtatacccat
     4381 acgatgttcc tgactatgcg ggctatccct atgacgtccc ggactatgca ggatcgtatc
     4441 cttatgacgt tccagattac gctggatccg ccgctccggc agctaagaaa aagaaactgg
     4501 atttcgaatc cggagaattc ggggcccagc cggccagatc tatggcggaa gcgggtatca
     4561 ccggcacgtg gtacaaccag tctggttcta ccttcaccgt taccgcgggt gcggacggta
     4621 acctgaccgg tcagtacgaa aaccgtgcgc agggcactgg ttgccagaac tctccgtaca
     4681 ccctgaccgg tcgttacaac ggtaccaaac tggaatggcg tgttgaatgg aacaactcta
     4741 ccgaaaactg ccactctcgt accgaatggc gtggtcagta ccagggtggt gcggaagcgc
     4801 gtatcaacac ccagtggaac ctgacctacg aaggtggttc tggtccggcg accgaacagg
     4861 gtcaggacac cttcaccaaa gttaaaccgt ctgcggcgtc tggatccccc gggggatcca
     4921 ctagttctag aggatccgcc gctcccgccg ccaagcgcgt gaagctggac ggcggcagcg
     4981 gcggcggcag cggcggcggc agcggctccg gaccggcagc taagagggtg aaactggatt
     5041 gataagcggc cgccaccgcg gtggagctcc agcttttgtt ccctttagtg agggttaatc
     5101 tcgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa
     5161 tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca
     5221 acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca
     5281 agtaaaacct ctacaaatgt ggtacttaag agggggagac caaagggcga gacgttaagg
     5341 cctcacgtga catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5401 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     5461 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     5521 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttacggg atacctgtcc gcctttctcc
     5581 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     5641 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     5701 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     5761 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     5821 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     5881 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     5941 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     6001 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     6061 ggattttggt catgccgtct cagaagaact cgtcaagaag gcgatagaag gcgatgcgct
     6121 gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg gtcagcccat tcgccgccaa
     6181 gctcttcagc aatatcacgg gtagccaacg ctatgtcctg atagcggtcc gccacaccca
     6241 gccggccaca gtcgatgaat ccagaaaagc ggccattttc caccatgata ttcggcaagc
     6301 aggcatcgcc atgggtcacg acgagatcct cgccgtcggg catgctcgcc ttgagcctgg
     6361 cgaacagttc ggctggcgcg agcccctgat gctcttcgtc cagatcatcc tgatcgacaa
     6421 gaccggcttc catccgagta cgtgctctct cgatgcgatg tttcgcttgg tggtcgaatg
     6481 ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc atcagccatg atggatactt
     6541 tctcggcagg agcaaggtga gatgacagga gatcctgccc cggcacttcg cccaatagca
     6601 gccagtccct tcccgcttca gtgacaacgt cgagtacagc tgcgcaagga acgcccgtcg
     6661 tggccagcca cgatagccgc gctgcctcgt cttgcagttc attcagggca ccggacaggt
     6721 cggtcttgac aaaaagaacc gggcgcccct gcgctgacag ccggaacacg gcggcatcag
     6781 agcagccgat tgtctgttgt gcccagtcat agccgaatag cctctccacc caagcggccg
     6841 gagaacctgc gtgcaatcca tcttgttcaa tcataatatt attgaagcat ttatcagggt
     6901 tcgtctcgtc ccggtctcct cccatgcatg
//
