LOCUS       sequence-90691-e        6807 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses truncated C-terminal SpCas9 domain fused to a GyrA intein,
            flanked by ITRs for AAV packaging. Combine with N-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 6807)
  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 6807)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: C-Terminal Split Cas9 with GyrA intein
FEATURES             Location/Qualifiers
     source          1..6807
                     /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..1004
                     /label=chicken beta-actin promoter
     intron          1005..1232
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1244..1253
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3626..3673
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(3701..3718)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3707..3914
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3923..4063
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3923..4052
                     /label=AAV2 ITR
     rep_origin      4138..4593
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4225..4244)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4435..4456
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4610..4629)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4729..4751
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4789..4807)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4875..4979
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4980..5048,5049..5840)
                     /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(5198..5217)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6011..6599
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6500..6519
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   6661..6801
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6672..6801
                     /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 ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga
      901 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
      961 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct
     1021 gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga
     1081 ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc
     1141 tgagcaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg tttaattacc
     1201 tggagcacct gcctgaaatc actttttttc aggttggacc ggtgccacca tgggaaaacc
     1261 ggaatttgcc ccaactacat atactgtcgg cgtcccaggg ctggttcgat tcctggaagc
     1321 ccaccacagg gatccagacg ctcaggcaat cgcagacgag ctgaccgacg ggaggtttta
     1381 ttatgctaag gtcgcctctg tgaccgatgc aggcgtacag cctgtgtatt ctcttagggt
     1441 ggacacagcc gaccatgctt ttattaccaa tggcttcgtt agtcacaata ccggctgggg
     1501 caggctgagc cggaagctga tcaacggcat ccgggacaag cagtccggca agacaatcct
     1561 ggatttcctg aagtccgacg gcttcgccaa cagaaacttc atgcagctga tccacgacga
     1621 cagcctgacc tttaaagagg acatccagaa agcccaggtg tccggccagg gcgatagcct
     1681 gcacgagcac attgccaatc tggccggcag ccccgccatt aagaagggca tcctgcagac
     1741 agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt
     1801 gatcgaaatg gccagagaga accagaccac ccagaaggga cagaagaaca gccgcgagag
     1861 aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc cagatcctga aagaacaccc
     1921 cgtggaaaac acccagctgc agaacgagaa gctgtacctg tactacctgc agaatgggcg
     1981 ggatatgtac gtggaccagg aactggacat caaccggctg tccgactacg atgtggacca
     2041 tatcgtgcct cagagctttc tgaaggacga ctccatcgac aacaaggtgc tgaccagaag
     2101 cgacaagaac cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa
     2161 gaactactgg cggcagctgc tgaacgccaa gctgattacc cagagaaagt tcgacaatct
     2221 gaccaaggcc gagagaggcg gcctgagcga actggataag gccggcttca tcaagagaca
     2281 gctggtggaa acccggcaga tcacaaagca cgtggcacag atcctggact cccggatgaa
     2341 cactaagtac gacgagaatg acaagctgat ccgggaagtg aaagtgatca ccctgaagtc
     2401 caagctggtg tccgatttcc ggaaggattt ccagttttac aaagtgcgcg agatcaacaa
     2461 ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa
     2521 gtaccctaag ctggaaagcg agttcgtgta cggcgactac aaggtgtacg acgtgcggaa
     2581 gatgatcgcc aagagcgagc aggaaatcgg caaggctacc gccaagtact tcttctacag
     2641 caacatcatg aactttttca agaccgagat taccctggcc aacggcgaga tccggaagcg
     2701 gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg gccgggattt
     2761 tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt
     2821 gcagacaggc ggcttcagca aagagtctat cctgcccaag aggaacagcg ataagctgat
     2881 cgccagaaag aaggactggg accctaagaa gtacggcggc ttcgacagcc ccaccgtggc
     2941 ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt
     3001 gaaagagctg ctggggatca ccatcatgga aagaagcagc ttcgagaaga atcccatcga
     3061 ctttctggaa gccaagggct acaaagaagt gaaaaaggac ctgatcatca agctgcctaa
     3121 gtactccctg ttcgagctgg aaaacggccg gaagagaatg ctggcctctg ccggcgaact
     3181 gcagaaggga aacgaactgg ccctgccctc caaatatgtg aacttcctgt acctggccag
     3241 ccactatgag aagctgaagg gctcccccga ggataatgag cagaaacagc tgtttgtgga
     3301 acagcacaag cactacctgg acgagatcat cgagcagatc agcgagttct ccaagagagt
     3361 gatcctggcc gacgctaatc tggacaaagt gctgtccgcc tacaacaagc accgggataa
     3421 gcccatcaga gagcaggccg agaatatcat ccacctgttt accctgacca atctgggagc
     3481 ccctgccgcc ttcaagtact ttgacaccac catcgaccgg aagaggtaca ccagcaccaa
     3541 agaggtgctg gacgccaccc tgatccacca gagcatcacc ggcctgtacg agacacggat
     3601 cgacctgtct cagctgggag gcgacaaaag gccggcggcc acgaaaaagg ccggccaggc
     3661 aaaaaagaaa aagtaagaat tcctagagct cgctgatcag cctcgactgt gccttctagt
     3721 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     3781 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     3841 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agagaatagc
     3901 aggcatgctg gggagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg
     3961 cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc
     4021 cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat
     4081 tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg
     4141 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     4201 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     4261 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     4321 ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat
     4381 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     4441 tcttgttcca aactggaaca acactcaacc ctatctcggg ctattctttt gatttataag
     4501 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     4561 cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct
     4621 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     4681 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     4741 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
     4801 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     4861 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     4921 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     4981 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     5041 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     5101 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     5161 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     5221 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     5281 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     5341 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     5401 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     5461 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     5521 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     5581 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     5641 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggaagcc
     5701 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     5761 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     5821 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     5881 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     5941 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     6001 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     6061 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     6121 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     6181 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     6241 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     6301 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     6361 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     6421 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     6481 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     6541 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     6601 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     6661 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
     6721 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
     6781 actccatcac taggggttcc tacatgt
//
