LOCUS       sequence-429503-        6220 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector to drive the expression of eGFP in genetically defined 
            cell populations under the control of the 4xBRE regulatory element 
            of SMAD1 transcription factor and miniXon splicing casette in vivo.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6220)
  AUTHORS   Okur Z, Schlauri N, Bitsikas V, Panopoulou M, Karmakar K, Schreiner 
            D, Scheiffele P
  TITLE     Control of neuronal excitation-inhibition balance by BMP-SMAD1 
            signaling
  JOURNAL   bioRxiv 2023
REFERENCE   2  (bases 1 to 6220)
  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: "bioRxiv 
            2023"
COMMENT     Sequence Label: pAAV-2xflox-BRX-eGFP-NLS
FEATURES             Location/Qualifiers
     source          1..6220
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..456
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(88..107)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     298..319
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(473..492)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     592..614
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(652..670)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        738..842
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(843..911,912..1703)
                     /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(1061..1080)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1874..2462
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2363..2382
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2524..2653
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     polyA_signal    2686..2734
                     /note="synthetic polyadenylation signal"
     misc_feature    2748..2839
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     2788..2807
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     promoter        2957..3153
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3004..3139
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     protein_bind    complement(3188..3221)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(3272..3305)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(3334..3354)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(3358..3378)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(3382..3402)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(join(3412..4122,4123..4125))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             complement(join(3412..4122,4123..4125))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3454..3475)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3803..3822
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4062..4083
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      4219..4228
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    4728..4761
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    4812..4845
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4870..5458
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4923..4943)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5341..5352)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    5490..5966
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(5689..5708)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   6006..6146
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6006..6135
                     /label=AAV2 ITR
ORIGIN
        1 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
       61 ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
      121 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
      181 gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc
      241 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
      301 gactcttgtt ccaaactgga acaacactca actctatctc gggctattct tttgatttat
      361 aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta
      421 acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct
      481 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
      541 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
      601 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
      661 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
      721 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
      781 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
      841 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
      901 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
      961 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     1021 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     1081 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     1141 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     1201 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     1261 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     1321 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     1381 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     1441 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     1501 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     1561 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     1621 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     1681 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     1741 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     1801 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     1861 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     1921 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     1981 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     2041 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     2101 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     2161 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     2221 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     2281 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     2341 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     2401 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     2461 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     2521 tgtcctgcag gcagctgcgc gctcgctcgc tcactgaggc cgcccgggcg tcgggcgacc
     2581 tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc
     2641 actaggggtt cctgcggccg cacgcgccgt acttggagcg gccgcaataa aatatcttta
     2701 ttttcattac atctgtgtgt tggttttttg tgtgaatcga tagtactaac atacgctctc
     2761 catcaaaaca aaacgaaaca aaacaaacta gcaaaatagg ctgtccccag tgcaagtgca
     2821 ggtgccagaa catttctcta tcgataggta ccgggcctgg cgtctaacgg tctgagggcc
     2881 tggcgtctaa cggtctgagg gcctggcgtc taacggtctg agggcctggc gtctaacggt
     2941 ctgaagatct gcgatctgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     3001 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatcg ctgactaatt
     3061 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
     3121 ggaggctttt ttggaggcct aggcttttgc aaaaagcttg gcattccggt agctggtcga
     3181 ctccggaata acttcgtata ggatacttta tacgaagtta tgcagaatgg tagctggatt
     3241 gtagctgcta ttagcaatat gaaacctctt aataacttcg tatagcatac attatacgaa
     3301 gttatggcgc gccgaatagg gccctctaga tcataccttt ctcttctttt ttggatctac
     3361 ctttctcttc ttttttggat ctacctttct cttctttttt ggatcggatc ccttgtacag
     3421 ctcgtccatg ccgagagtga tcccggcggc ggtcacgaac tccagcagga ccatgtgatc
     3481 gcgcttctcg ttggggtctt tgctcagggc ggactgggtg ctcaggtagt ggttgtcggg
     3541 cagcagcacg gggccgtcgc cgatgggggt gttctgctgg tagtggtcgg cgagctgcac
     3601 gctgccgtcc tcgatgttgt ggcggatctt gaagttcacc ttgatgccgt tcttctgctt
     3661 gtcggccatg atatagacgt tgtggctgtt gtagttgtac tccagcttgt gccccaggat
     3721 gttgccgtcc tccttgaagt cgatgccctt cagctcgatg cggttcacca gggtgtcgcc
     3781 ctcgaacttc acctcggcgc gggtcttgta gttgccgtcg tccttgaaga agatggtgcg
     3841 ctcctggacg tagccttcgg gcatggcgga cttgaagaag tcgtgctgct tcatgtggtc
     3901 ggggtagcgg ctgaagcact gcacgccgta ggtcagggtg gtcacgaggg tgggccaggg
     3961 cacgggcagc ttgccggtgg tgcagatgaa cttcagggtc agcttgccgt aggtggcatc
     4021 gccctcgccc tcgccggaca cgctgaactt gtggccgttt acgtcgccgt ccagctcgac
     4081 caggatgggc accaccccgg tgaacagctc ctcgcccttg ctcacagatc ctcttaccca
     4141 cgggaaacaa caatttcttg ttgtttggtt cctagatcac caaaaaataa agatcataaa
     4201 caaccagaac ccagcctggc caccatggtg aaaccccgtc tctactaaaa atacaaaaat
     4261 tagccaggcg tgaatgctgt gtggtcagtt aggctgcctc tcagtaccac attaagtatt
     4321 cagtaccaca ttaagtattc agagttaaac ctcaacaaaa ttatgtctta ctctacacaa
     4381 tcatgaacct cctgcatggt ggcacttgga ttgactcaaa aatgttaaat ctgcaaatgg
     4441 caagaaaaag tttttatttt ataaacatta gtacctcact ttcagtaact ttgtatttcc
     4501 taatccaata tttgaaattt cacacaattc ctgttttgtc ataaatggaa aacatttcct
     4561 gcacaagaga ctaagcaaga tatgtattaa ttcacttaaa atttcaaaag atggtaacga
     4621 gagaacttac cagaaaagtt tccaagaatg gcaaagctga tgccagtggc tctctgcaag
     4681 gttaagttgt acagaaattt accaacagta ccggattgcc agctagcata acttcgtata
     4741 aagtatccta tacgaagtta tttgccttaa cccagaaatt atcactgtta ttctttagaa
     4801 tggtgcaaag aataacttcg tataatgtat gctatacgaa gttatgaatt cgatatcaag
     4861 cttatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac
     4921 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt
     4981 gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat
     5041 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca
     5101 acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc
     5161 cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
     5221 gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct
     5281 tggctgctcg cctatgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct
     5341 tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt
     5401 ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat
     5461 cgataccgag cgctgctcga gagatctacg ggtggcatcc ctgtgacccc tccccagtgc
     5521 ctctcctggc cctggaagtt gccactccag tgcccaccag ccttgtccta ataaaattaa
     5581 gttgcatcat tttgtctgac taggtgtcct tctataatat tatggggtgg aggggggtgg
     5641 tatggagcaa ggggcaagtt gggaagacaa cctgtagggc ctgcggggtc tattgggaac
     5701 caagctggag tgcagtggca caatcttggc tcactgcaat ctccgcctcc tgggttcaag
     5761 cgattctcct gcctcagcct cccgagttgt tgggattcca ggcatgcatg accaggctca
     5821 gctaattttt gtttttttgg tagagacggg gtttcaccat attggccagg ctggtctcca
     5881 actcctaatc tcaggtgatc tacccacctt ggcctcccaa attgctggga ttacaggcgt
     5941 gaaccactgc tcccttccct gtccttctga ttttgtaggt aaccacgtgc ggaccgagcg
     6001 gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     6061 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     6121 cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct
     6181 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt
//
