LOCUS       sequence-395623-        7745 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses AAV2 Rep and AAV9 Cap, with GFP replacing a region of Cap,
            within AAV2 ITRs..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7745)
  AUTHORS   Gonzalez TJ, Mitchell-Dick A, Blondel LO, Fanous MM, Hull JA, Oh DK,
            Moller-Tank S, Castellanos Rivera RM, Piedrahita JA, Asokan A
  TITLE     Structure-guided AAV capsid evolution strategies for enhanced CNS 
            gene delivery.
  JOURNAL   Nat Protoc. 2023 Sep 21. doi: 10.1038/s41596-023-00875-y.
  PUBMED    37735235
REFERENCE   2  (bases 1 to 7745)
  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: "Nat Protoc.
            2023 Sep 21. doi: 10.1038/s41596-023-00875-y."
COMMENT     Sequence Label: pITR-AAV-Rep2Cap9-GFP
FEATURES             Location/Qualifiers
     source          1..7745
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(3256..3258,3259..3261,3262..3975)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3256..3258,3259..3261,3262..3972)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3301..3322)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3562..3581)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3909..3930
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     repeat_region   4029..4158
                     /label=AAV2 ITR
     rep_origin      4529..5042
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4616..4635)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4826..4847
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5523..5542)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5642..5664
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5702..5720)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5788..5892
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5893..5961,5962..6753)
                     /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(6111..6130)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6924..7512
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7413..7432
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cagctgcgcg ctcgctcgct cactgaggcc gcccgggcgt cgggcgacct ttggtcgccc
       61 ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc aactccatca ctaggggttc
      121 cttgtagtta atgattaacc cgccatgcta cttatctacg tagccatgct ctagctagag
      181 gtcctgtatt agaggtcacg tgagtgtttt gcgacatttt gcgacaccat gtggtcacgc
      241 tgggtattta agcccgagtg agcacgcagg gtctccattt tgaagcggga ggtttgaacg
      301 cgcagccgcc atgccggggt tttacgagat tgtgattaag gtccccagcg accttgacga
      361 gcatctgccc ggcatttctg acagctttgt gaactgggtg gccgagaagg aatgggagtt
      421 gccgccagat tctgacatgg atctgaatct gattgagcag gcacccctga ccgtggccga
      481 gaagctgcag cgcgactttc tgacggaatg gcgccgtgtg agtaaggccc cggaggccct
      541 tttctttgtg caatttgaga agggagagag ctacttccac atgcacgtgc tcgtggaaac
      601 caccggggtg aaatccatgg ttttgggacg tttcctgagt cagattcgcg aaaaactgat
      661 tcagagaatt taccgcggga tcgagccgac tttgccaaac tggttcgcgg tcacaaagac
      721 cagaaatggc gccggaggcg ggaacaaggt ggtggatgag tgctacatcc ccaattactt
      781 gctccccaaa acccagcctg agctccagtg ggcgtggact aatatggaac agtatttaag
      841 cgcctgtttg aatctcacgg agcgtaaacg gttggtggcg cagcatctga cgcacgtgtc
      901 gcagacgcag gagcagaaca aagagaatca gaatcccaat tctgatgcgc cggtgatcag
      961 atcaaaaact tcagccaggt acatggagct ggtcgggtgg ctcgtggaca aggggattac
     1021 ctcggagaag cagtggatcc aggaggacca ggcctcatac atctccttca atgcggcctc
     1081 caactcgcgg tcccaaatca aggctgcctt ggacaatgcg ggaaagatta tgagcctgac
     1141 taaaaccgcc cccgactacc tggtgggcca gcagcccgtg gaggacattt ccagcaatcg
     1201 gatttataaa attttggaac taaacgggta cgatccccaa tatgcggctt ccgtctttct
     1261 gggatgggcc acgaaaaagt tcggcaagag gaacaccatc tggctgtttg ggcctgcaac
     1321 taccgggaag accaacatcg cggaggccat agcccacact gtgcccttct acgggtgcgt
     1381 aaactggacc aatgagaact ttcccttcaa cgactgtgtc gacaagatgg tgatctggtg
     1441 ggaggagggg aagatgaccg ccaaggtcgt ggagtcggcc aaagccattc tcggaggaag
     1501 caaggtgcgc gtggaccaga aatgcaagtc ctcggcccag atagacccga ctcccgtgat
     1561 cgtcacctcc aacaccaaca tgtgcgccgt gattgacggg aactcaacga ccttcgaaca
     1621 ccagcagccg ttgcaagacc ggatgttcaa atttgaactc acccgccgtc tggatcatga
     1681 ctttgggaag gtcaccaagc aggaagtcaa agactttttc cggtgggcaa aggatcacgt
     1741 ggttgaggtg gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa gacccgcccc
     1801 cagtgacgca gatataagtg agcccaaacg ggtgcgcgag tcagttgcgc agccatcgac
     1861 gtcagacgcg gaagcttcga tcaactacgc agacaggtac caaaacaaat gttctcgtca
     1921 cgtgggcatg aatctgatgc tgtttccctg cagacaatgc gagagaatga atcagaattc
     1981 aaatatctgc ttcactcacg gacagaaaga ctgtttagag tgctttcccg tgtcagaatc
     2041 tcaacccgtt tctgtcgtca aaaaggcgta tcagaaactg tgctacattc atcatatcat
     2101 gggaaaggtg ccagacgctt gcactgcctg cgatctggtc aatgtggatt tggatgactg
     2161 catctttgaa caataaatga tttaaatcag gtatggctgc cgatggttat cttccagatt
     2221 ggctcgagga caaccttagt gaaggaattc gcgagtggtg ggctttgaaa cctggagccc
     2281 ctcaacccaa ggcaaatcaa caacatcaag acaacgctcg aggtcttgtg cttccgggtt
     2341 acaaatacct tggacccggc aacggactcg acaaggggga gccggtcaac gcagcagacg
     2401 cggcggccct cgagcacgac aaggcctacg accagcagct caaggccgga gacaacccgt
     2461 acctcaagta caaccacgcc gacgccgagt tccaggagcg gctcaaagaa gatacgtctt
     2521 ttgggggcaa cctcgggcga gcagtcttcc aggccaaaaa gaggcttctt gaacctcttg
     2581 gtctggttga ggaagcggct aagacggctc ctggaaagaa gaggcctgta gagcagtctc
     2641 ctcaggaacc ggactcctcc gcgggtattg gcaaatcggg tgcacagccc gctaaaaaga
     2701 gactcaattt cggtcagact ggcgacacag agtcagtccc agaccctcaa ccaatcggag
     2761 aacctcccgc agccccctca ggtgtgggat ctcttacaat ggcttcaggt ggtggcgcac
     2821 cagtggcaga caataacgaa ggtgccgatg gagtgggtag ttcctcggga aattggcatt
     2881 gcgattccca atggctgggg gacagagtca tcaccaccag cacccgaacc tgggccctgc
     2941 ccacctacaa caatcacctc tacaagcaaa tctccaacag cacatctgga ggatcttcaa
     3001 atgacaacgc ctacttcggc tacagcaccc cctgggggta ttttgacttc aacagattcc
     3061 actgccactt ctcaccacgt gactggcagc gactcatcaa caacaactgg ggattccggc
     3121 ctaagcgact caacttcaag ctcttcaaca ttcaggtcaa agaggttacg gacaacaatg
     3181 gagtcaagac catcgccaat aaccttacca gcacggtcca ggtcttcacg gactcagact
     3241 atcagctccc gtacgatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     3301 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     3361 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     3421 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac
     3481 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     3541 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     3601 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     3661 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
     3721 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
     3781 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     3841 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
     3901 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     3961 gagctgtaca agtagtctag agcatggcta cgtagataag tagcatggcg ggttaatcat
     4021 taactacaag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct
     4081 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt
     4141 gagcgagcga gcgcgcagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca
     4201 acagttgcgc agcctgaatg gcgaatggcg attccgttgc aatggctggc ggtaatattg
     4261 ttctggatat taccagcaag gccgatagtt tgagttcttc tactcaggca agtgatgtta
     4321 ttactaatca aagaagtatt gcgacaacgg ttaatttgcg tgatggacag actcttttac
     4381 tcggtggcct cactgattat aaaaacactt ctcaggattc tggcgtaccg ttcctgtcta
     4441 aaatcccttt aatcggcctc ctgtttagct cccgctctga ttctaacgag gaaagcacgt
     4501 tatacgtgct cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
     4561 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     4621 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     4681 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     4741 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     4801 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     4861 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     4921 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     4981 atttaaaaat ttgcttatac aatcttcctg tttttggggc ttttctgatt atcaaccggg
     5041 gtacatatga ttgacatgct agttttacga ttaccgtcat cgattctctt gtttgctcca
     5101 gactctcagg caatgacctg atagcctttg tagagacctc tcaaaaatag ctaccctctc
     5161 cggcatgaat ttatcagcta gaacggttga atatcatatt gatggtgatt tgactgtctc
     5221 cggcctttct cacccgtttg aatctttacc tacacattac tcaggcattg catttaaaat
     5281 atatgagggt tctaaaaatt tttatccttg cgttgaaata aaggcttctc ccgcaaaagt
     5341 attacagggt cataatgttt ttggtacaac cgatttagct ttatgctctg aggctttatt
     5401 gcttaatttt gctaattctt tgccttgcct gtatgattta ttggatgttg gaatcgcctg
     5461 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc
     5521 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     5581 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     5641 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     5701 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     5761 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     5821 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     5881 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     5941 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     6001 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     6061 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     6121 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     6181 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     6241 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     6301 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     6361 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     6421 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     6481 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     6541 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     6601 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     6661 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     6721 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     6781 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     6841 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     6901 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     6961 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     7021 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     7081 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     7141 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     7201 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     7261 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     7321 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     7381 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     7441 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     7501 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     7561 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     7621 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     7681 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     7741 taatg
//
