LOCUS       sequence-346968-        7395 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  In vitro transcription of SauCas9 mRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7395)
  AUTHORS   Takasugi PR, Wang S, Truong KT, Drage EP, Kanishka SN, Higbee MA, 
            Bamidele N, Ojelabi O, Sontheimer EJ, Gagnon JA
  TITLE     Orthogonal CRISPR-Cas tools for genome editing, inhibition, and 
            CRISPR recording in zebrafish embryos.
  JOURNAL   Genetics. 2021 Nov 4. pii: 6420709. doi: 10.1093/genetics/iyab196.
  PUBMED    34735006
REFERENCE   2  (bases 1 to 7395)
  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: "Genetics. 
            2021 Nov 4. pii: 6420709. doi: 10.1093/genetics/iyab196."
COMMENT     Sequence Label: pCS2-SauCas9
FEATURES             Location/Qualifiers
     source          1..7395
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      605..1060
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(692..711)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     902..923
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1086..1190
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1191..1259,1260..2051)
                     /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(1409..1428)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2222..2810
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2711..2730
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2964..2981
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3098..3119
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3134..3139,3140..3157,3158..3164)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3172..3188
                     /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)."
     primer_bind     3177..3199
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3196..3212
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3196..3212
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3231..3251
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3233..3251
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     polyA_signal    3364..3498
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3401..3420)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3455..3474
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(3531..3557)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(3558..3584)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(3591..3638)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(3639..6794)
                     /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             complement(6819..6839)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(6892..6910)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(6893..6910)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        complement(join(6945..7395,1..534))
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 accatatata gagaccattg acgtcaatgg gggagtggct atgggcggta ttaggaagcc
       61 ccatatatgg tatatgggac cgcccattgg gaggggctat ctacgtcaat aggaaaaccc
      121 atatatggaa tactatatgg catagggcca atacatagta ttgaacctgg ccaatagcca
      181 tattggcata gggccatatt ggatattgcc tatatattga tcctggcata tagccaatat
      241 ggccgccatt attggcacca tgccaattca atatggcgga cttggcacag ggccaagacc
      301 cctcctcaaa cttggcaccg tgccaagtag acccctcccc agttggcaca gtgccaggtc
      361 cgccatattg aattggcatg gtgccaagtc cgccatattg aattggctgg ggtccagatc
      421 caccatattg aattggcacg agtccatata cgccatattg aattggcaca ggtccagatc
      481 caccatattg aattggcatg agtccatata cgccatattg aattggcggt cgactggcgt
      541 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
      601 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
      661 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
      721 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
      781 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
      841 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
      901 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
      961 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     1021 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa
     1081 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     1141 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     1201 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     1261 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     1321 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     1381 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     1441 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     1501 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     1561 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     1621 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     1681 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     1741 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     1801 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     1861 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     1921 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     1981 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     2041 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     2101 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     2161 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     2221 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     2281 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     2341 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     2401 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     2461 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     2521 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     2581 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     2641 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     2701 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     2761 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     2821 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     2881 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     2941 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
     3001 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     3061 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt
     3121 aggcacccca ggctttacac tttatgctcc cggctcgtat gttgtgtgga attgtgagcg
     3181 gataacaatt tcacacagga aacagctatg accatgatta cgccaagcgc gcaattaacc
     3241 ctcactaaag ggaacaaaag ctgggtaccg ggcccaatgc attggcgccg cggccgcgaa
     3301 ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt
     3361 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     3421 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     3481 tcttatcatg tctggatcta cgtaatacga ctcactatag ttctagatta agcgtaatct
     3541 ggaacatcgt atgggtaagc gtaatctgga acatcgtatg ggtaggatcc ctttttcttt
     3601 tttgcctggc cggccttttt cgtggccgcc ggccttttgc cctttttgat gatctgaggg
     3661 tgcttcttag atttcacttc atacaggttg cccagaatgt ctgtgctgta cttcttaatg
     3721 ctctgggtct tggaggcgat tgtcttaatg atcctggggg gcctcttgtc gttcatgttt
     3781 tccaggtact cgcggtaggt gatgtcgatc atgttcactt cgatccggtt cagcaggtcg
     3841 ttgttcacgc cgatcactct atacagctcg ccgttgatct tgatcagatc gttgttgtag
     3901 aaggaggcga taaactcggc ctggttgctg atcttcttca gcttcttagc ttcctcatag
     3961 cacttgctat tcacttcgta gtagttttct tttttgatca catccagatt cttcacggtc
     4021 acgaacttgt acacgccatt gtccaggtac acgtcgaatc tgtagggctt cagggacagc
     4081 ttcacgacct tgtttctgct gttggggtag tcgtcggtga tgtccagatg ggcgttcagt
     4141 ttgttgccgt aatacttaat cttcttgatc acggggccgt tgtccttttt ggagtacttg
     4201 gtcaggtagt tcccggtttc ctcgtagtac ttgtacaggg gattcttctc gtcgccgtac
     4261 tgttccataa tcagcttcag tttctggtag gtctgggggt cgtggtggta catcagcagc
     4321 ttttcggggc tcttgttgat cagctttttc agcttgtcat tgtccttgtc gtacaggccg
     4381 ttcagattgt tcacgatcag ggtgttgccc ttgtcgtcct tccgggtgga gtacagggtg
     4441 tcgttaatca gctctctatt aggcttcttg tccacccggt ggctgtactt gtagtccttg
     4501 aagtccttaa tgtgcttgat ctggtggggg gtgatgaaga tctctttgta ctcctgctcg
     4561 gtttcgatct cgggcatgct ctcggcctgc ttttcctcga acatctggtt ttccatcact
     4621 tttttggcct tgtccagttt cttccactct ttgaagatga aatcggcgtt ggcaatgatc
     4681 agggcgtcct cggcgtggtg cttgtacccc ttgttccgct ctttcttaaa cttccacttc
     4741 cgccgcagaa agctggtgaa gccgccattg atggacttca ctttcacgtc caggttgttc
     4801 actctgaagt agctccgcag caggttcatc aggcctctgg tggcgtatct ggtatccacc
     4861 aggttccggt tgatgaagtc tttctgcacg gagaacctgt tgatgtcccg ttcttccagc
     4921 agatactctt tcttggtctt gctgattctg cccttgccct tggccagatt caggatgtgc
     4981 ttcttgaagg tttcgtagct gatcttgctg tcgctgctgc tcaggtactg gaatggggtc
     5041 cggttgccct tcttgctgtt ttcttcctgc ttcacgagca ccttgttgtt gaagctgttg
     5101 tcgaaggaca cgcttctggg gatgatgtgg tccacctcat agttgaaggg gttgttcagc
     5161 agatcttcca gagggatggc ttccaggctg tacaggcact tgccttcctg catgtcgtgc
     5221 agcttgatct tctcgatcag gtacttggcg ttctctttgc cggtggtccg gatgatttcc
     5281 tcgatccgct cgttggtctg ccggttccgc ttctgcatct cgttgatcat tttctgggcg
     5341 tccttggagt tcttctcgcg ggccagctcg ataatgatgt cgttgggcag gccgtacttc
     5401 ttgatgatgg cgttgatcac tttgatgctc tggatgaagc ttctcttcac gacggggctc
     5461 aggatgaagt cgtccaccag ggtggtgggg atctctttct gctgggacag gtccaccttc
     5521 ttgggcacca gcttcagccg gttgaagata gcgatctggt tgtcgttggt gtgccacagc
     5581 tcgtccagga tcaggttgat ggccttcagg ctcaggttgt gggtgccggt atagcccttc
     5641 agattagaga tctgctcgat ctcttcctgg gtcagctcgg agttcagatt ggtcagttct
     5701 tcctggatgt cctcgctgct ctggtagatg gtcaggatct tggcaatctg atccagcagc
     5761 tcggcgttct caataatctc tttccgggcg gtaatgtcct tgatgtcgtg gtacaccttc
     5821 aggttggtga actcgggctt gccggtgctg gtcactctgt agcccttaat atcctcttcg
     5881 ttcacgagga tttctttggc gatctgcttc agggtgggct tcttcttctg cttgaacacg
     5941 ttctcgatga tctggaactt ctcgtaatat tccagcttct cgttctcgtc cctggtgatc
     6001 acgagattgt tcaggtcgtt cagggcgttg tacaggtcgg cgttgtaggc gtacttcacg
     6061 ctccgcagtt cctcggggaa gtaggtgcag tggcccatca gcatctcgta ccattctttg
     6121 atgtccttcc agccgaaggg gctgccctcg ccaggtccct catagtaggt ccgccgggtt
     6181 tccagcaggt cgatgtaggt gtcgatgaag ctctggtcca gctggtggta ggccttctgc
     6241 accttcagca gctgtttggc ttctttcacg tagtcgctgg tcttgaatct gttgatgctg
     6301 ccccgcactt cgccgtcttt cttcagccgt tccagctgca gttcggccac gtatttctct
     6361 tccagggcct tgctgttccg gctgatctgc tctttggtgg acagctcgtt gccggtgtcc
     6421 tcttccacct cgttcacgtt gtgcacgcct cttctcttgg ccaggtgcag cagggcggca
     6481 gagaactctt cctcgctcag cttctggctc aggcccttca ctctggcctc gtaggggttg
     6541 atgccgctca gctcgctgtg gtcggtcagc aggttgtagt cgaacagcag cttcttcact
     6601 ctctggattc tatgccgcct ccgccgcttc agccttctgg cgcctctctt gctccgcctg
     6661 ccctcgttgt tttccacgtt ggcctctttg aacagccgca cgccggcatc gatcacgtcc
     6721 cgtgtctcgt agtcgatgat gccgtagccc acgctggtga tgccgatgtc caggcccagg
     6781 atgtagttcc gcttggctgc tgggactccg tggataccga ccttccgctt cttctttggg
     6841 gccatgaatt cgaatcgatg ggatcctgca aaaagaacaa gtagcttgta ttctatagtg
     6901 tcacctaaat caagcttgct ccgacgtccc caggcagaat ggcggttccc taaacgagca
     6961 ttgcttatat agacctccca ttaggcacgc ctaccgccca tttacgtcaa tggaacgccc
     7021 atttgcgtca ttgcccctcc ccattgacgt caatggggat gtacttggca gccatcgcgg
     7081 gccatttacc gccattgacg tcaatgggag tactgccaat gtaccctggc gtacttccaa
     7141 tagtaatgta cttgccaagt tactattaat agatattgat gtactgccaa gtgggccatt
     7201 taccgtcatt gacgtcaata gggggcgtga gaacggatat gaatgggcaa tgagccatcc
     7261 cattgacgtc aatggtgggt ggtcctattg acgtcaatgg gcattgagcc aggcgggcca
     7321 tttaccgtaa ttgacgtcaa tgggggaggc gccatatacg tcaataggac cgcccatatg
     7381 acgtcaatag gaaag
//
