LOCUS       sequence-174242-        7122 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gene correction donor AAV for RCS rat. AAV backbone plasmid 
            including exon 2 of rat Mertk and rMertkgRNA for HITI.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7122)
  AUTHORS   Suzuki K, Tsunekawa Y, Hernandez-Benitez R, Wu J, Zhu J, Kim EJ, 
            Hatanaka F, Yamamoto M, Araoka T, Li Z, Kurita M, Hishida T, Li M, 
            Aizawa E, Guo S, Chen S, Goebl A, Soligalla RD, Qu J, Jiang T, Fu X,
            Jafari M, Esteban CR, Berggren WT, Lajara J, Nunez-Delicado E, 
            Guillen P, Campistol JM, Matsuzaki F, Liu GH, Magistretti P, Zhang 
            K, Callaway EM, Zhang K, Belmonte JC
  TITLE     In vivo genome editing via CRISPR/Cas9 mediated homology-independent
            targeted integration.
  JOURNAL   Nature. 2016 Dec 1;540(7631):144-149. doi: 10.1038/nature20565. Epub
            2016 Nov 16.
  PUBMED    27851729
REFERENCE   2  (bases 1 to 7122)
  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"; doi: 
            "10.1038/nature20565"; journalName: "Nature"; date: "2016-12-1- 1"; 
            volume: "540"; issue: "7631"; pages: "144-149"
COMMENT     Sequence Label: pAAV-rMERTK-HITI
FEATURES             Location/Qualifiers
     source          1..7122
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        160..408
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     160..180
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     331..350
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        429..504
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(516..540)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        2802..3013
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             3026..3294
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      3318..3327
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3324..3326,3327..3329,3330..4040)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3324..3326,3327..3329,3330..4040)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3369..3390)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3630..3649)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3977..3998
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4314..4362
                     /note="synthetic polyadenylation signal"
     repeat_region   4385..4525
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4385..4514
                     /label=AAV2 ITR
     rep_origin      4600..5055
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4687..4706)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4897..4918
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5072..5091)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5191..5213
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5251..5269)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5337..5441
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5442..5510,5511..6302)
                     /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(5660..5679)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6473..7061
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6962..6981
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtaaggtc gggcaggaag agggcctatt tcccatgatt
      181 ccttcatatt tgcatatacg atacaaggct gttagagaga taattagaat taatttgact
      241 gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag
      301 tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag
      361 tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccga ggaccactgc
      421 aacggggcgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt
      481 gaaaaagtgg caccgagtcg gtgctttttt tctagaccca gctttcttgt acaaagttgg
      541 cgtttaaacc cagccccgtt gcagtggtcc tcctgccttg ctcaccgcct caaccattca
      601 ctgccaacat atttgaaaga acagcaacca gcagagaatg tgtgttccca agtaccgctt
      661 acacaagctc aagttcagtg tatatgagtc tctattttta gaaatacttg cagaccagat
      721 aaaataaaaa catttcctgc ctcgttcatg tagacatgca tttgctagaa ggtccagatc
      781 tggagcctaa gagaactttc cctgttggac acaaaacttc taaacccatg tctttgtgac
      841 ccacagtggc cattctggtt gtctgactga acagatgggg agtaggaaag gtgaccctag
      901 accctcatat agctgtcact ttcctgctag gttatgtata tagaacttgg agtgagttac
      961 cacttgacaa aaattgtcag agaaacttcg ttgtttaaaa tttatatact tggtgtgtat
     1021 ataggaggtg gtgcacatcc ttctgtacac atgcggaggt cagaggacac cttgcaggag
     1081 tcagttctct cctcccactg tgtgggctct ggagatggag ctcagggtca tcaggctggg
     1141 cagtaagcac ctcttaccca ctgagccatc tcaccagccc aggaaattaa cttccttcct
     1201 tccttccttc cttccttcct tccttccttc cttccttcct tctttcttcg tttttttttt
     1261 ttcccagaga gggtttttct atgtaataga gccctgtcta tcctgtactc gctttgtaga
     1321 tcaggctggc cttgaactca cagagatcca actgcctctg tctcctgagt attgggattg
     1381 aaggcaagca ccaccacacc aggccagaat tttcttaagt attctttaga acagtggttc
     1441 gaaaccttct taatgctgtg accatttggt acagttcctc atattgtgat gactcccaac
     1501 cataaaatgt tttcattgct acttcataac tgtggttatt ctgctactgt tatgaatcat
     1561 aatgtaaata tttttggaaa tagagctgcc aacgtggagg agacctacag gttgagaagc
     1621 acccgggtac acaaattgtt aactgaggat agtcgtcaga tgtgcgtatc tgtcaggaag
     1681 ttctccccgc ctcttttctc tttgttaagg tgctaggaac tgaactgggg gactcgtgtg
     1741 ggtgaggcag tgttctacca ctgagctaca tcccagctct tgattctttt gggacagatt
     1801 ttcactgtgt aactcaggtt ggtcttgagc tctcggccct cctgcctcag cctcacagat
     1861 gcttggatga caagctacgc ctttgcatcc aacttcgtgc ttatcttctc tgaggccacg
     1921 gaggatgttc tgcttcaccg actggtgtgg tgtcagcatc tgaagttttt ctccatcatc
     1981 ttaaaggcta aaactctact attttgcttt gcaggcactg ctgaaaagga ggaagaaatc
     2041 aagccagatc agccattttc agggccttta ccagggagcc taccagctga ccacaggcca
     2101 ttcttcgccc ctcactccag tggggaccag ctgtcaccat cccagactgg aagatcacat
     2161 ccagcacaca cagccactcc ccagatgacc tctgcagcat caaacctcct gcctcctgtt
     2221 gcatttaaaa acacaattgg acgcatagta ctttcggaac ataaaagtgt caaatttaat
     2281 tgctcgatca acattcctaa cgtgtatcaa gaaacagctg gcatttcgtg gtggaaagat
     2341 ggaaaggaac tgcttggggc acatcattca atcacacagt tttatcctga tgaggaaggg
     2401 gtatcaataa ttgcattgtt caggtatgtg tccttccttt ctaaatgcca tcgtgctaat
     2461 accccctcca aagcagaatc cccaaggaaa gagcaccctg gttttgtatg tatgattgtt
     2521 cgtcaagcaa tcttgatcct tttagagggt agatacatag ctgacctggc tgtgttgttg
     2581 atttctttta aatgagcttg tattgtggta actgctggct aacacacgct ctgagggttt
     2641 agagggctgc tcccagctca tctaaggtga gtattaagta caccttagat ccaatggcta
     2701 ccagagagtg ggaaacagag tgtatgaagg gggtgtgttc atgtttgtgc ctgtgcgtgt
     2761 gtgcgccagc cccgttgcag tggtcctcgc tagctgtcag tgggcagagc gcacatcgcc
     2821 cacagtcccc gagaagttgg ggggaggggt cggcaattga acgggtgcct agagaaggtg
     2881 gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg
     2941 gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc
     3001 cgccagaaca cagctgaagc ttcgaggggc tcgcatctct ccttcacgcg cccgccgccc
     3061 tacctgaggc cgccatccac gccggttgag tcgcgttctg ccgcctcccg cctgtggtgc
     3121 ctcctgaact gcgtccgccg tctaggtaag tttaaagctc aggtcgagac cgggcctttg
     3181 tccggcgctc ccttggagcc tacctagact cagccggctc tccacgcttt gcctgaccct
     3241 gcttgctcaa ctctacgtct ttgtttcgtt ttctgttctg cgccgttaca gatccaagct
     3301 gtgaccggcg cgtcgacgcc accatggtga gcaagggcga ggagctgttc accggggtgg
     3361 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     3421 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     3481 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
     3541 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     3601 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     3661 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     3721 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     3781 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     3841 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     3901 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca
     3961 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     4021 gcatggacga gctgtacaag tccggactca gatctcgaga ggaggaggag gagacagaca
     4081 gcaggatgcc ccacctcgac agccccggca gctcccagcc gagacgctcc ttcctctcaa
     4141 gggtgatcag ggcagcgcta ccgttgcagc tgcttctgct gctgctgctg ctcctggcct
     4201 gcctgctacc tgcctctgaa gatgactaca gctgcaccca ggccaacaac tttgcccgat
     4261 ccttctaccc catgctgcgg tacaccaacg ggccacctcc cacctaggaa ttcaataaaa
     4321 gatctttatt ttcattagat ctgtgtgttg gttttttgtg tgcacgtgcg gaccgagcgg
     4381 ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact
     4441 gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc
     4501 gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg
     4561 tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat
     4621 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccttag
     4681 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     4741 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     4801 ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     4861 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     4921 caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg
     4981 tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     5041 taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     5101 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     5161 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     5221 cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta
     5281 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     5341 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     5401 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     5461 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     5521 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     5581 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     5641 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     5701 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     5761 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     5821 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     5881 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     5941 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     6001 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     6061 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     6121 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     6181 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     6241 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     6301 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     6361 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     6421 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     6481 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     6541 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     6601 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     6661 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     6721 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     6781 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     6841 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     6901 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     6961 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     7021 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     7081 ttttacggtt cctggccttt tgctggcctt ttgctcacat gt
//
