LOCUS       sequence-308662-        7332 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7332)
  AUTHORS   Jao LE, Wente SR, Chen W
  TITLE     Efficient multiplex biallelic zebrafish genome editing using a 
            CRISPR nuclease system.
  JOURNAL   Proc Natl Acad Sci U S A. 2013 Aug 5.
  PUBMED    23918387
REFERENCE   2  (bases 1 to 7332)
  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: "Proc Natl 
            Acad Sci U S A. 2013 Aug 5."
COMMENT     Sequence Label: pT3TS-nCas9n
FEATURES             Location/Qualifiers
     source          1..7332
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     5'UTR           44..86
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     regulatory      102..111
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             120..140
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             147..4247
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes MGAS15252 Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFGSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLADSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQIYNQLFEENPINASRVDAKAILSARLSKSRRL
                     ENLIAQLPGEKRNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNSEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGAYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRGMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGHSLHEQIANLAGSPAIKKGILQTVKIVDELVKVMGH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     RBS             4160..4168
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             4263..4283
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     3'UTR           4308..4433
                     /label=Xenopus globin 3'-UTR
                     /note="3'-UTR of the major beta-globin gene of Xenopus 
                     laevis"
     primer_bind     complement(4520..4539)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4521..4539)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4546..4563)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4546..4562)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4555..4577)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(4704..5159)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4841..4862)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5053..5072
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5186..5290
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5291..5359,5360..6151)
                     /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(5509..5528)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6322..6910
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6811..6830
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7064..7081
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7198..7219
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7234..7239,7240..7257,7258..7264)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7272..7288
                     /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     7277..7299
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7296..7312
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7296..7312
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 aattaaccct cactaaaggg aacaaaagct ggagctaagc ttgcttgttc tttttgcaga
       61 agctcagaat aaacgctcaa ctttggcaga tctaactcga cgccaccatg gcttctccac
      121 ctaagaagaa gagaaaggtg ggaagcatgg ataagaagta tagcatcggc ctggatattg
      181 gaactaactc cgtgggttgg gcagtgatta cagacgacta caaggtccct agcaagaaat
      241 ttaaggtgct gggtaacacc gacaggcaca gcatcaagaa aaatctgatt ggagccctgc
      301 tgttcggttc tggagagact gccgaagcaa cacgcctgaa aagaacagca agaaggcgct
      361 ataccagaag gaagaataga atctgttacc tgcaggagat tttctctaac gaaatggcta
      421 aggtggacga ttcattcttt cataggctgg aggaaagttt cctggtcgag gaagataaga
      481 aacacgagcg ccatcctatc tttggaaaca ttgtggacga ggtcgcctat cacgaaaaat
      541 acccaaccat ctatcatctg cgcaagaaac tggctgactc tactgataaa gccgacctga
      601 gactgatcta tctggctctg gcccacatga ttaagttcag gggtcatttt ctgatcgagg
      661 gcgatctgaa ccccgacaat tccgatgtgg acaagctgtt catccagctg gtccagattt
      721 acaatcagct gtttgaggaa aaccctatta atgcttccag agtggacgca aaagctatcc
      781 tgtcagccag gctgtccaag tcacgcagac tggagaacct gattgcacag ctgcccggag
      841 aaaagaggaa cggtctgttt ggaaatctga tcgctctgag tctgggcctg actcctaact
      901 tcaaaagcaa ttttgatctg gctgaggacg ccaaactgca gctgtcaaag gacacatatg
      961 acgatgacct ggataacctg ctggcacaga tcggagatca gtacgctgac ctgttcctgg
     1021 ctgccaaaaa tctgtccgac gcaatcctgc tgtcagatat tctgagagtg aacagcgaga
     1081 ttacaaaagc tcctctgagt gccagcatga tcaagagata tgacgagcac catcaggatc
     1141 tgaccctgct gaaggctctg gtcaggcagc agctgccaga gaagtacaag gaaattttct
     1201 ttgatcagtc caagaacggc tacgccggtt atatcgacgg aggcgcatca caggaggaat
     1261 tctacaagtt tatcaaacct attctggaga agatggacgg aactgaggaa ctgctggtga
     1321 aactgaatag agaggacctg ctgaggaagc agcgcacatt tgataacggt tccatcccac
     1381 accagattca tctgggagag ctgcacgcta tcctgaggcg ccaggaagac ttctacccct
     1441 ttctgaaaga taaccgcgag aagatcgaaa aaattctgac cttcagaatc ccttactatg
     1501 tgggtccact ggctcgcgga aacagcagat ttgcctggat gactcgcaaa tccgaggaaa
     1561 ccattactcc ttggaacttc gaggaagtgg tcgataaggg cgcctctgca cagtccttca
     1621 tcgagagaat gactaatttt gacaaaaacc tgcccaatga gaaagtgctg cctaagcact
     1681 ccctgctgta cgagtatttc actgtctata acgaactgac aaaggtgaaa tacgtcaccg
     1741 agggcatgag aaagccagcc ttcctgtcag gagagcagaa gaaagcaatc gtggatctgc
     1801 tgtttaaaac caacaggaaa gtgactgtca agcagctgaa ggaggactac ttcaagaaaa
     1861 ttgaatgctt cgattccgtg gagatcagcg gagtcgaaga cagatttaac gcaagcctgg
     1921 gcgcttacca cgatctgctg aagatcatta aggataaaga cttcctggac aacgaggaaa
     1981 atgaggatat cctggaagac attgtgctga cactgaccct gtttgaggac agaggaatga
     2041 tcgaggaaag actgaaaacc tatgctcatc tgttcgatga caaggtgatg aaacagctga
     2101 agagaaggcg ctacactggc tggggtagac tgagcaggaa gctgatcaac ggcattaggg
     2161 ataaacagtc aggaaagaca atcctggact ttctgaaaag tgatggcttc gccaaccgca
     2221 attttatgca gctgattcac gatgacagtc tgaccttcaa agaggacatc cagaaggctc
     2281 aggtgtctgg acagggccac tccctgcatg agcagattgc aaacctggct ggaagcccag
     2341 ccatcaagaa aggcattctg cagacagtga aaatcgtcga tgagctggtg aaagtcatgg
     2401 gccataagcc cgaaaacatc gtgattgaga tggctcgcga aaatcagaca acccagaagg
     2461 gtcagaagaa cagtagagag aggatgaaaa gaatcgagga aggcattaag gagctgggta
     2521 gccagatcct gaaagagcac ccagtggaaa acacacagct gcagaatgag aagctgtatc
     2581 tgtactatct gcagaatgga agagatatgt acgtggacca ggagctggat attaacaggc
     2641 tgtctgatta cgacgtggat catatcgtcc cccagagttt catcaaagat gacagcattg
     2701 acaacaaggt gctgaccagg tccgacaaaa acagaggaaa atcagataat gtccctagtg
     2761 aggaagtggt caagaaaatg aagaactact ggagacagct gctgaatgcc aaactgatca
     2821 ctcagaggaa gtttgataac ctgacaaaag cagagcgcgg tggactgtca gaactggaca
     2881 aagctggatt catcaagagg cagctggtgg aaacacgcca gatcactaaa cacgtcgcac
     2941 agattctgga tagtcgcatg aacacaaagt acgatgagaa tgacaaactg atcagagaag
     3001 tgaaggtcat taccctgaag agtaaactgg tcagcgactt taggaaagat ttccagtttt
     3061 ataaggtccg cgagattaac aattatcacc atgcccatga cgcatacctg aacgccgtgg
     3121 tcggtaccgc actgatcaag aaatacccaa aactggagag cgaattcgtg tacggagact
     3181 ataaggtgta cgatgtcaga aaaatgatcg ccaagtccga gcaggaaatt ggaaaagcta
     3241 ctgccaagta tttcttttac tcaaacatca tgaatttctt taagacagag atcaccctgg
     3301 ccaatggaga aatccgcaaa aggcccctga ttgagacaaa cggagagaca ggcgaaatcg
     3361 tgtgggacaa aggcagagat tttgcaaccg tgaggaaggt cctgagcatg cctcaagtga
     3421 atatcgtcaa gaaaactgag gtgcagacag gcggtttctc aaaagaaagt attctgccaa
     3481 aacgcaactc tgataagctg atcgctagaa agaaagactg ggaccctaag aagtatggag
     3541 gctttgactc tcccactgtg gcatactccg tcctggtggt cgctaaggtg gagaagggca
     3601 aaagcaagaa actgaaatct gtcaaggagc tgctgggtat cacaattatg gagagaagct
     3661 ctttcgagaa gaacccaatc gattttctgg aggccaaagg ttataaggaa gtgaagaaag
     3721 acctgatcat taaactgccc aagtacagtc tgtttgagct ggaaaacggc aggaaacgca
     3781 tgctggcaag cgctggagag ctgcagaaag gcaatgaact ggccctgcct tctaagtacg
     3841 tgaacttcct gtatctggca agccactacg agaagctgaa aggatctcca gaggataacg
     3901 aacagaaaca gctgtttgtg gagcagcaca agcattatct ggacgagatc attgaacaga
     3961 ttagcgagtt ctctaaaaga gtgatcctgg ccgacgcaaa tctggataag gtcctgtctg
     4021 cttacaacaa acacagagat aagcccatca gggagcaggc cgaaaatatc attcatctgt
     4081 tcactctgac aaacctgggc gcacctgcag ctttcaagta cttcgacact acaatcgata
     4141 gaaagaggta cacctccact aaggaggtgc tggacgctac actgatccat cagagtatta
     4201 ccggcctgta cgaaacaagg attgacctgt ctcagctggg tggcgactca ccggtgagat
     4261 ctcctaagaa gaagagaaag gtgtgaccgc ggatctggtt accactaaac cagcctcaag
     4321 aacacccgaa tggagtctct aagctacata ataccaactt acactttaca aaatgttgtc
     4381 ccccaaaatg tagccattcg tatctgctcc taataaaaag aaagtttctt cacattctaa
     4441 aaaaaaaaaa aaaaaaaaaa aaaaaaancc cccccccccc cctgcagggc caagtcggcc
     4501 tctagaggta cccaattcgc cctatagtga gtcgtattac aattcactgg ccgtcgtttt
     4561 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     4621 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     4681 gcgcagcctg aatggcgaat ggcaaattgt aagcgttaat attttgttaa aattcgcgtt
     4741 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     4801 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     4861 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     4921 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     4981 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     5041 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     5101 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     5161 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     5221 ttcaaatatg tatctgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     5281 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     5341 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     5401 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     5461 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     5521 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     5581 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     5641 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     5701 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     5761 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     5821 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     5881 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     5941 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     6001 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     6061 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     6121 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     6181 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     6241 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     6301 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     6361 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     6421 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     6481 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     6541 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     6601 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     6661 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     6721 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     6781 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     6841 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     6901 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     6961 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     7021 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     7081 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     7141 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     7201 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
     7261 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
     7321 cgccaagctc ga
//
