LOCUS       sequence-492918-        6824 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses truncated N-terminal SpCas9 domain fused to a GyrA intein,
            flanked by ITRs for AAV packaging. Combine with C-Terminal Split 
            Cas9 Gyra Intein for full length SpCas9 production.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6824)
  AUTHORS   Fine EJ, Appleton CM, White DE, Brown MT, Deshmukh H, Kemp ML, Bao G
  TITLE     Trans-spliced Cas9 allows cleavage of HBB and CCR5 genes in human 
            cells using compact expression cassettes.
  JOURNAL   Sci Rep. 2015 Jul 1;5:10777. doi: 10.1038/srep10777.
  PUBMED    26126518
REFERENCE   2  (bases 1 to 6824)
  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: "Sci Rep."; 
            date: "2015-07-1"; volume: "5:10777. doi"; pages: " 
            10.1038/srep10777"
COMMENT     Sequence Label: N-Terminal Split Cas9 with GyrA intein
FEATURES             Location/Qualifiers
     source          1..6824
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        440..725
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        727..1003
                     /label=chicken beta-actin promoter
     intron          1004..1231
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1243..1252
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1252..1317
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1324..1344
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1369..3084
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     primer_bind     complement(3718..3735)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3724..3931
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3940..4080
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3940..4069
                     /label=AAV2 ITR
     rep_origin      4155..4610
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4242..4261)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4452..4473
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4627..4646)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4746..4768
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4806..4824)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4892..4996
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4997..5065,5066..5857)
                     /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(5215..5234)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6028..6616
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6517..6536
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   6678..6818
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6689..6818
                     /label=AAV2 ITR
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
      421 aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      481 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc
      541 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      601 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt
      661 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      721 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac
      781 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg
      841 gggggggcgc gcgccaggcg gggcggggcg gggcgagggg cggggcgggg cgaggcggag
      901 aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg
      961 gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg
     1021 ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac
     1081 cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagct
     1141 gagcaagagg taagggttta agggatggtt ggttggtggg gtattaatgt ttaattacct
     1201 ggagcacctg cctgaaatca ctttttttca ggttggaccg gtgccaccat ggactataag
     1261 gaccacgacg gagactacaa ggatcatgat attgattaca aagacgatga cgataagatg
     1321 gccccaaaga agaagcggaa ggtcggtatc cacggagtcc cagcagccga caagaagtac
     1381 agcatcggcc tggacatcgg caccaactct gtgggctggg ccgtgatcac cgacgagtac
     1441 aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg accggcacag catcaagaag
     1501 aacctgatcg gagccctgct gttcgacagc ggcgaaacag ccgaggccac ccggctgaag
     1561 agaaccgcca gaagaagata caccagacgg aagaaccgga tctgctatct gcaagagatc
     1621 ttcagcaacg agatggccaa ggtggacgac agcttcttcc acagactgga agagtccttc
     1681 ctggtggaag aggataagaa gcacgagcgg caccccatct tcggcaacat cgtggacgag
     1741 gtggcctacc acgagaagta ccccaccatc taccacctga gaaagaaact ggtggacagc
     1801 accgacaagg ccgacctgcg gctgatctat ctggccctgg cccacatgat caagttccgg
     1861 ggccacttcc tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga caagctgttc
     1921 atccagctgg tgcagaccta caaccagctg ttcgaggaaa accccatcaa cgccagcggc
     1981 gtggacgcca aggccatcct gtctgccaga ctgagcaaga gcagacggct ggaaaatctg
     2041 atcgcccagc tgcccggcga gaagaagaat ggcctgttcg gaaacctgat tgccctgagc
     2101 ctgggcctga cccccaactt caagagcaac ttcgacctgg ccgaggatgc caaactgcag
     2161 ctgagcaagg acacctacga cgacgacctg gacaacctgc tggcccagat cggcgaccag
     2221 tacgccgacc tgtttctggc cgccaagaac ctgtccgacg ccatcctgct gagcgacatc
     2281 ctgagagtga acaccgagat caccaaggcc cccctgagcg cctctatgat caagagatac
     2341 gacgagcacc accaggacct gaccctgctg aaagctctcg tgcggcagca gctgcctgag
     2401 aagtacaaag agattttctt cgaccagagc aagaacggct acgccggcta cattgacggc
     2461 ggagccagcc aggaagagtt ctacaagttc atcaagccca tcctggaaaa gatggacggc
     2521 accgaggaac tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca gcggaccttc
     2581 gacaacggca gcatccccca ccagatccac ctgggagagc tgcacgccat tctgcggcgg
     2641 caggaagatt tttacccatt cctgaaggac aaccgggaaa agatcgagaa gatcctgacc
     2701 ttccgcatcc cctactacgt gggccctctg gccaggggaa acagcagatt cgcctggatg
     2761 accagaaaga gcgaggaaac catcaccccc tggaacttcg aggaagtggt ggacaagggc
     2821 gcttccgccc agagcttcat cgagcggatg accaacttcg ataagaacct gcccaacgag
     2881 aaggtgctgc ccaagcacag cctgctgtac gagtacttca ccgtgtataa cgagctgacc
     2941 aaagtgaaat acgtgaccga gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa
     3001 aaggccatcg tggacctgct gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa
     3061 gaggactact tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg cgtggaagat
     3121 cggttcaacg cctccctggg cacataccac gatctgctga aaattatcaa ggacaaggac
     3181 ttcctggaca atgaggaaaa cgaggacatt ctggaagata tcgtgctgac cctgacactg
     3241 tttgaggaca gagagatgat cgaggaacgg ctgaaaacct atgcccacct gttcgacgac
     3301 aaagtgatga agcagctgaa gcggcggaga tactgtatca ccggtgatgc tctggtggcg
     3361 ctgcccgaag gcgaatcagt gcgcatcgct gacatcgtcc cgggcgcccg ccctaatagc
     3421 gataacgcca tcgacctgaa ggtgcttgac cgccacggca atcctgttct ggccgataga
     3481 ctcttccaca gtggtgaaca tcctgtgtat accgtccgca ccgtagaagg cctgagagtg
     3541 acaggcacag ccaaccaccc ccttttgtgt ctggtggatg ttgccggcgt ccccaccctg
     3601 ctgtggaaac tgattgatga gataaaaccc ggagactatg cagtgatcca aaggtccgca
     3661 tttagtgtgg attgcgctgg cttcgctcgc taagaattcc tagagctcgc tgatcagcct
     3721 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     3781 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     3841 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     3901 attgggaaga gaatagcagg catgctgggg agcggccgca ggaaccccta gtgatggagt
     3961 tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc
     4021 gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg
     4081 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc
     4141 aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     4201 gcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc
     4261 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     4321 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg
     4381 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     4441 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta
     4501 ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat
     4561 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac
     4621 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     4681 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     4741 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
     4801 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     4861 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     4921 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     4981 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     5041 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     5101 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     5161 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     5221 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     5281 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     5341 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     5401 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     5461 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     5521 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     5581 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     5641 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     5701 cggtgagcgt ggaagccgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     5761 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     5821 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     5881 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     5941 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     6001 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     6061 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     6121 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     6181 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     6241 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     6301 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     6361 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     6421 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     6481 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     6541 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     6601 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     6661 gccttttgct cacatgtcct gcaggcagct gcgcgctcgc tcgctcactg aggccgcccg
     6721 ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg
     6781 cagagaggga gtggccaact ccatcactag gggttcctac atgt
//
