LOCUS       sequence-176253-        4583 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV-gRNA cloning vector with GFP reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4583)
  AUTHORS   Jarrett KE, Lee CM, Yeh YH, Hsu RH, Gupta R, Zhang M, Rodriguez PJ, 
            Lee CS, Gillard BK, Bissig KD, Pownall HJ, Martin JF, Bao G, Lagor 
            WR
  TITLE     Somatic genome editing with CRISPR/Cas9 generates and corrects a 
            metabolic disease.
  JOURNAL   Sci Rep. 2017 Mar 16;7:44624. doi: 10.1038/srep44624.
  PUBMED    28300165
REFERENCE   2  (bases 1 to 4583)
  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: "2017-03-16"; volume: "7:44624. doi"; pages: " 
            10.1038/srep44624"
COMMENT     Sequence Label: 1179_pAAV-U6-BbsI-gRNA-CB-EmGFP
FEATURES             Location/Qualifiers
     source          1..4583
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   4..133
                     /label=AAV2 ITR
     promoter        255..503
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     255..275
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     426..445
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        522..597
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             join(1078..1080,1081..1083,1084..1797)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1123..1144)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1384..1403)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1731..1752
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1848..1896
                     /note="synthetic polyadenylation signal"
     repeat_region   1944..2073
                     /label=AAV2 ITR
     rep_origin      complement(2310..2898)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2390..2409)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3069..3860,3861..3929))
                     /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     3692..3711
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3930..4034)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4060..4515)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4197..4218)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4409..4428
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cagctgcgcg ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc
       61 tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc
      121 actaggggtt ccttgtagtt aatgattaac ccgccatgct acttatctac gtagccatgc
      181 tctggtgtac aaaaaagcag gctttaaagg aaccaattca gtcgactgga tccggtacca
      241 aggtcgggca ggaagagggc ctatttccca tgattccttc atatttgcat atacgataca
      301 aggctgttag agagataatt agaattaatt tgactgtaaa cacaaagata ttagtacaaa
      361 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
      421 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
      481 tcttgtggaa aggacgaaac accgggtctt cgagaagacc tgttttagag ctagaaatag
      541 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
      601 ttttctagac ccagctttca agcttgatta acccgccatg ctacttatct acgtagccat
      661 gctctaggaa gatcggaatt cgcccttaag ctagtatgcc aagtacgccc cctattgacg
      721 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
      781 ctacttggca gtacatctac tcgaggccac gttctgcttc actctcccca tctccccccc
      841 ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag cgatgggggc
      901 gaggggcggg gcggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc
      961 cgaaagtttc cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg
     1021 cggcgggcgg gagcgggatc agaatgatct gatatcatcg atgaattcga gctcaccatg
     1081 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     1141 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     1201 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     1261 gtgaccacct tcacctacgg cgtgcagtgc ttcgcccgct accccgacca catgaagcag
     1321 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     1381 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     1441 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     1501 ctggagtaca actacaacag ccacaaggtc tatatcaccg ccgacaagca gaagaacggc
     1561 atcaaggtga acttcaagac ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     1621 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     1681 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     1741 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaagaa
     1801 ttctgcagtc gacgcgggcc cgggatccac cggtgcggcc gctcgcgaat aaaagatctt
     1861 tattttcatt agatctgtgt gttggttttt tgtgtgatgc agctacgtag ataagtagca
     1921 tggcgggtta atcattaact acaaggaacc cctagtgatg gagttggcca ctccctctct
     1981 gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc
     2041 ccgggcggcc tcagtgagcg agcgagcgcg cagctgcatt aatgaatcgg ccaacgcgcg
     2101 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     2161 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     2221 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     2281 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     2341 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     2401 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     2461 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     2521 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     2581 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     2641 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     2701 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     2761 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     2821 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     2881 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     2941 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     3001 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     3061 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     3121 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     3181 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     3241 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     3301 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     3361 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     3421 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     3481 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     3541 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     3601 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     3661 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     3721 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     3781 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     3841 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     3901 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     3961 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     4021 ataggggttc cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc gttaatattt
     4081 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     4141 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     4201 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     4261 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     4321 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     4381 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     4441 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     4501 cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg
     4561 tgcgggcctc ttcgctatta cgc
//
