LOCUS       sequence-462041-        5973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An adeno-associated viral vector to express a SpCas9 guide RNA 
            targeting rat beta actin also carrying an HDR template for insertion
            of mEGFP to the break site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5973)
  TITLE     Ribeiro Gomes plasmids 2024
REFERENCE   2  (bases 1 to 5973)
  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"
COMMENT     Sequence Label: pOTTC2360 - pAAV rActin EGFP donor
FEATURES             Location/Qualifiers
     source          1..5973
                     /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        149..397
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     149..169
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     320..339
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        419..494
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             join(1490..1492,1493..1495,1496..1498,1499..2209)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MHVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT
                     LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKD
                     DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGI
                     KVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVL
                     LEFVTAAGITLGMDELYK"
     primer_bind     complement(1538..1559)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1799..1818)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2146..2167
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             complement(2676..2687)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   3236..3376
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3236..3365
                     /label=AAV2 ITR
     rep_origin      3451..3906
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3538..3557)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3748..3769
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3923..3942)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4042..4064
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4102..4120)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4188..4292
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4293..4361,4362..5153)
                     /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(4511..4530)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5324..5912
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5813..5832
                     /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 gcggcctcta gaaagcttga gggcctattt cccatgattc cttcatattt
      181 gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa
      241 gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt
      301 aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt
      361 cttggcttta tatatcttgt ggaaaggacg aaacaccgtg tgccttgata gttcgccagt
      421 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg
      481 caccgagtcg gtgctttttt tgaattctgc agctcctccg tcccggtcca cacccgccac
      541 caggtaagca gggacgtcgg gcccagcggg ccccaacttt accttggcca ctacctcgct
      601 gcaggatcgt gaggaacact cagaagggac accgtagagg ggtggagcgt ggtaccgggc
      661 cgcggagcgg acactggcaa agcttaactt tccgcctagg gtgtagagtg tttgcagtcg
      721 tattcccgcg gtgtagacac tcgtgggcac gctcctgctt ggtgcgcggg gcttggggac
      781 acactagagt cgcggtgtgg gcatttggag agccggtgcg gcttgcgggt gttaagccgc
      841 atctgtccac cttgagggga cacagtattg ggagtcaggc gttacaatca cgctttgatg
      901 gcctatgggt ctttgtccaa accggttttg cccattcggc ttggcgggcg cggcggggcc
      961 ggctcggccg ggtgggggct gggatgccat tgcgcgtgcg cgctctatca ctgggcattg
     1021 gggcgcgtgc gcgctgggga gggaactctt cctctccccc tcttccgagt taagagttgc
     1081 gcgtgcgtat tgagactagg agcgcggccg ccccgggttg ggcgagggcg gggccgttgc
     1141 ccggaagggg cggggtcgta gcggctaggg cgcctgctcg cgcttcctgc tgggtgtggt
     1201 cgcctcccgc gcgcgcacta gccgcccgtc gcctcagtgt aggcggggcc tgtgcccgtt
     1261 tggggagggg gcggaggcct ggcttcctgc cgtgggtccg cctccgggcc agcgtttgcc
     1321 ttttatggta ataatgcggc tgtcctgcgc ttcctttgtc ccctgagctt gggcgcgcgc
     1381 cccctggcgg ctcgaggccg cggcttgccg gaagtgggca gggcggcagc ggctgctctt
     1441 ggcggctccg cggtgaccat agccctcttt tgtgccttga tagttcgcca tgcatgtgag
     1501 caagggcgag gagctcttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
     1561 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     1621 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     1681 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
     1741 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     1801 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     1861 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     1921 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     1981 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     2041 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     2101 cacccagtcc aagctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     2161 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagg atgacgatat
     2221 cgctgcgctc gtcgtcgaca acggctccgg catgtgcaag gccggcttcg cgggcgacga
     2281 tgctccccgg gccgtcttcc cctccatcgt gggccgccct aggcaccagg taagtgaccc
     2341 tttactttgg gagtggcagc cctagggttt tcttgggggt cgatgccagt gctgagaacg
     2401 ttgttctcct ccgcagggtg tgatggtggg tatgggtcag aaggactcct acgtgggcga
     2461 cgaggcccag agcaagagag gcatcctgac cctgaagtac cccattgaac acggcattgt
     2521 caccaactgg gacgatatgg agaagatttg gcaccacact ttctacaatg agctgcgtgt
     2581 ggcccctgag gagcaccctg tgctgctcac cgaggcccct ctgaacccta aggccaaccg
     2641 tgaaaagatg acccaggtca gtatcctggg tgacccctcc cctctattgg gccaacttct
     2701 cagccacgcc ctttctcaat tgtctttctt ctgccattct cccataggac tctcttctat
     2761 gagctgagtc tcccttggaa ctttgcagtt tctgctcttt cccagatgag gtcttttttt
     2821 tctctcgatc gcctttctga ctaggtgttt taaaccccta cagtgctgtg ggtgtaggta
     2881 ctaacaatgg ctcgtgtgac aaagctaatg aggctggtga taaatggcct tggagtgtgt
     2941 attcagtaga tgcacagtag gtctaaatgg agcccctgtc ctgatactcc cagcacactt
     3001 aacttagctg tgttcttgcc ctctttgcat gtctcactca aatctatcct tacagtctca
     3061 cctgccctga gtgtttcttg tggctttagg agcttgacaa tactgtattc ctttctctac
     3121 agatcatgtt tgagaccttc aacaccccag ccatgtacgt agccatccag gctgtgttgt
     3181 ccctgtatgc ctctggtcgt accactggca ttgtgatgga cggatccgcg gccgcaggaa
     3241 cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg
     3301 cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg
     3361 cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     3421 tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg
     3481 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc
     3541 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     3601 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     3661 ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     3721 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     3781 actctatctc gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt
     3841 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta
     3901 caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     3961 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     4021 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     4081 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     4141 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     4201 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     4261 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     4321 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     4381 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     4441 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     4501 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     4561 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     4621 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     4681 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     4741 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     4801 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     4861 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     4921 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     4981 ctgataaatc tggagccggt gagcgtggaa gccgcggtat cattgcagca ctggggccag
     5041 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     5101 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     5161 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     5221 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     5281 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     5341 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     5401 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     5461 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     5521 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     5581 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     5641 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     5701 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     5761 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     5821 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     5881 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     5941 tcctggcctt ttgctggcct tttgctcaca tgt
//
