LOCUS       sequence-200006-        7161 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV expressing membrane-targeted SNAP protein and nuclear-targeted 
            eGFP after FLP-mediated recombination.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7161)
  AUTHORS   Ciabatti E, Gonzalez-Rueda A, Mariotti L, Morgese F, Tripodi M
  TITLE     Life-Long Genetic and Functional Access to Neural Circuits Using 
            Self-Inactivating Rabies Virus.
  JOURNAL   Cell. 2017 Jul 1. pii: S0092-8674(17)30697-9. doi: 
            10.1016/j.cell.2017.06.014.
  PUBMED    28689641
REFERENCE   2  (bases 1 to 7161)
  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: "Cell. 2017 
            Jul 1. pii: S0092-8674(17)30697-9. doi: 10.1016/j.cell.2017.06.014."
COMMENT     Sequence Label: pAAV-CMV-FRT-myrSNAP-2A-H2BeGFP-WPRE
FEATURES             Location/Qualifiers
     source          1..7161
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     178..197
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     437..458
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(585..962)
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSAK"
     CDS             complement(981..1034)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(1044..1589)
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="fast-reacting variant, optimized for mammalian 
                     expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVE
                     VPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISYSHLAALAGNPAATAAVKTALSGNPVPILIPCHRVVQGDLDVGGYEGGLA
                     VKEWLLAHEGHRLGKPGLG"
     regulatory      1649..1658
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(1730..1763)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     protein_bind    complement(1774..1807)
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     misc_feature    1831..2419
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1884..1904)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2302..2313)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2454..2471)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2460..2667
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2690..2830
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2690..2819
                     /label=AAV2 ITR
     rep_origin      2905..3360
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2992..3011)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3202..3223
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3377..3396)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3496..3518
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3556..3574)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3642..3746
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3747..3815,3816..4607)
                     /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(3965..3984)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4778..5366
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5267..5286
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5428..5557
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        5633..5936
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5937..6140
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6091..6111
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     6137..6161
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          6289..6667
                     /label=chimeric intron
                     /note="fusion between human cytomegalovirus intron A and 
                     human beta-globin intron 2"
     protein_bind    6767..6800
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     protein_bind    6811..6844
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     CDS             complement(join(6948..7161,1..497,498..500))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(6990..7011)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 atcttgaagt tggccttgat gccgttcttc tgcttgtcgg ccatgatata gacgttgtgg
       61 ctgttgtagt tgtactccag cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg
      121 cccttcagct cgatgcggtt caccagggtg tcgccctcga acttcacctc ggcgcgggtc
      181 ttgtagttgc cgtcgtcctt gaagaagatg gtgcgctcct ggacgtagcc ttcgggcatg
      241 gcggacttga agaagtcgtg ctgcttcatg tggtcggggt agcggctgaa gcactgcacg
      301 ccgtaggtca gggtggtcac gagggtgggc cagggcacgg gcagcttgcc ggtggtgcag
      361 atgaacttca gggtcagctt gccgtaggtg gcatcgccct cgccctcgcc ggacacgctg
      421 aacttgtggc cgtttacgtc gccgtccagc tcgaccagga tgggcaccac cccggtgaac
      481 agctcctcgc ccttgctcat ggtggcccgc ggtagcgcta gcccacctcc gccgctgccc
      541 ccacctccag atccaccccc gccggagcct ccacccccag atcccttagc gctggtgtac
      601 ttggtgacgg ccttagtacc ctcggacacg gcgtgcttgg ccaactcccc aggcagcagc
      661 aggcgcacgg ccgtctggat ctccctggag gtgatggtcg agcgcttgtt gtaatgcgcc
      721 aggcgggaag cctcacctgc gatgcgctcg aaaatgtcgt tcacaaacga gttcatgatg
      781 cccatggcct tggacgaaat gccggtgtca gggtggacct gcttcagaac cttgtacaca
      841 tagatggaat agctctcctt gcggctgcgc ttgcgcttct tgccgccttt cttctgcgcc
      901 ttagtcaccg ccttcttgga gccctttttc ggggcgggag cagacttcgc tggctctggc
      961 atggtgatat tcagatctcc tgggccagga ttctcctcga cgtcaccgca tgttagcaga
     1021 cttcctctgc cctctccact gccacccagc ccaggcttgc ccagtctgtg gccctcgtgg
     1081 gccagcagcc actctttcac ggcgagcccg ccctcgtagc cccccacgtc caggtcgccc
     1141 tgcaccaccc ggtggcaggg gatcagaatg ggcacgggat ttccgctcag ggcggttttc
     1201 acggcggcgg tggcggcggg attgccggcc agggcggcca ggtggctgta gctgatgacc
     1261 tctccgaact tcaccacttt cagcagtttc cacagcacct ggcgggtaaa gctctcctgc
     1321 tggaacactg ggtggtgcag ggctggcaca gggaactcct cgatggcctc aggctggtga
     1381 aagtaggcgt tgagccaggc ggtggcctgc atcagtggct ctggtccgcc cagcacggcg
     1441 gctggggcag gcacttccac ggcgtcggcg gcagatgttc ctttgcccag gaagatgata
     1501 cggtgcaggc cctgttcgca cccagacagt tccagcttgc ccagagggct atccagggtg
     1561 gtgcgcttca tttcgcagtc tttgtccatg gaggcgaccg gtgcctcgtc gtcgttcagg
     1621 ttgtccttgc gcttgctctt gatgcagccc atggcggcct cgtcgtcgtt caggttgtcc
     1681 ttgcgcttgc tcttgatgca gcccatggtg ggaattgcgg ccggccttgg aagttcctat
     1741 actttctaga gaataggaac ttcccgaatc gcagaagttc ctatactatt tgaagaatag
     1801 gaacttccga agatcatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa
     1861 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
     1921 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
     1981 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
     2041 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
     2101 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
     2161 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg
     2221 gggaaatcat cgtcctttcc ttggctgctc gcctatgttg ccacctggat tctgcgcggg
     2281 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg
     2341 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc
     2401 ctttgggccg cctccccgca tcgataccga gcgctgctcg agagatcgat ctgcctcgac
     2461 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     2521 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     2581 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     2641 ggaagacaat agcaggcatg ctggggacac gtgcggaccg agcggccgca ggaaccccta
     2701 gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca
     2761 aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc
     2821 tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     2881 cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     2941 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg
     3001 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     3061 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     3121 tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     3181 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta
     3241 tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa
     3301 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
     3361 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc
     3421 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     3481 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     3541 gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     3601 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     3661 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     3721 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     3781 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     3841 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     3901 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     3961 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     4021 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     4081 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     4141 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     4201 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     4261 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     4321 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     4381 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     4441 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     4501 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     4561 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     4621 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     4681 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     4741 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     4801 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     4861 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     4921 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     4981 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     5041 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     5101 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     5161 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     5221 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     5281 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     5341 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     5401 ccttttgctg gccttttgct cacatgtcct gcaggcagct gcgcgctcgc tcgctcactg
     5461 aggccgcccg ggcgtcgggc gacctttggt cgcccggcct cagtgagcga gcgagcgcgc
     5521 agagagggag tggccaactc catcactagg ggttcctgcg gccgcacgcg tctagttatt
     5581 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
     5641 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
     5701 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
     5761 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
     5821 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
     5881 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga
     5941 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa
     6001 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc
     6061 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg
     6121 aggtctatat aagcagagct cgtttagtga accgtcagat cgcctggaga cgccatccac
     6181 gctgttttga cctccataga agacaccggg accgatccag cctccgcgga ttcgaatccc
     6241 ggccgggaac ggtgcattgg aacgcggatt ccccgtgcca agagtgacgt aagtaccgcc
     6301 tatagagtct ataggcccac aaaaaatgct ttcttctttt aatatacttt tttgtttatc
     6361 ttatttctaa tactttccct aatctctttc tttcagggca ataatgatac aatgtatcat
     6421 gcctctttgc accattctaa agaataacag tgataatttc tgggttaagg caatagcaat
     6481 atttctgcat ataaatattt ctgcatataa attgtaactg atgtaagagg tttcatattg
     6541 ctaatagcag ctacaatcca gctaccattc tgcttttatt ttatggttgg gataaggctg
     6601 gattattctg agtccaagct aggccctttt gctaatcatg ttcatacctc ttatcttcct
     6661 cccacagctc ctgggcaacg tgctggtctg tgtgctggcc catcactttg gcaaagaatt
     6721 gggattcgaa catcgattga attcggtacc cttcaggtcg acgtcggaag ttcctattct
     6781 ctagaaagta taggaacttc ccgaatcgca gaagttccta ttcttcaaat agtataggaa
     6841 cttcttggcg cgccgctagc ttcctcgagg caagctttca ctattactta ctatcaccta
     6901 ttatcacggc cgcccggatc cgatgagctt gatgatctga gtccggactt gtacagctcg
     6961 tccatgccgt gagtgatccc ggcggcggtc acgaactcca gcaggaccat gtgatcgcgc
     7021 ttctcgttgg ggtctttgct cagggcggac tgggtgctca ggtagtggtt gtcgggcagc
     7081 agcacggggc cgtcgccgat gggggtgttc tgctggtagt ggtcggcgag ctgcacgctg
     7141 ccgtcctcga tgttgtggcg g
//
