LOCUS       sequence-469419-        7304 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV2 vector containing EGFP and FlipO sequences separated by T2A 
            peptide under the control of CaMKIIA promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7304)
  AUTHORS   Baccini G, Foggetti A, Wernet N, Engelhardt KA, Kronenbitter K, 
            Michels J, Kulik A, Wozny C, Wulff P
  TITLE     Parvalbumin expression identifies subicular principal cells with 
            high projection specificity
  JOURNAL   bioRxiv 2024.08.13.606964
REFERENCE   2  (bases 1 to 7304)
  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 
            2024.08.13.606964"
COMMENT     Sequence Label: pAM-CaMKII-EGFP-T2A-FlipO
FEATURES             Location/Qualifiers
     source          1..7304
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   7..136
                     /label=AAV2 ITR
     promoter        191..1479
                     /label=CaMKII promoter
                     /note="promoter for the mouse calcium/calmodulin-dependent 
                     kinase II alpha subunit"
     CDS             join(1500..1502,1503..1505,1506..2216)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1500..1502,1503..1505,1506..2216)
                     /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(1545..1566)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1806..1825)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2153..2174
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2226..2279
                     /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             join(2289..2291,2292..2312,2313..3584)
                     /codon_start=1
                     /product="nuclear-targeted site-specific recombinase"
                     /label=FLPo
                     /note="FLPo is a mouse codon-optimized fusion between the 
                     SV40 nuclear localization signal and a thermostable version
                     of the Saccharomyces cerevisiae site-specific recombinase 
                     FLP.  Recombination occurs at FRT sequences."
                     /translation="APKKKRKVMSQFDILCKTPPKVLVRQFVERFERPSGEKIASCAAE
                     LTYLCWMITHNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKKL
                     IPAWEFTIIPYNGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESIW
                     EITEKILNSFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGVI
                     IQCLVTETKTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEYQ
                     LLKDNLVRSYNKALKKNAPYPIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWSD
                     KRASAVARTTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQLK
                     GSAEGSIRYPAWNGIISQEVLDYLSSYINRRI"
     misc_feature    3612..4200
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3665..3685)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4083..4094)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4227..4244)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4233..4457
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(4760..5348)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4840..4859)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5519..6310,6311..6379))
                     /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     6142..6161
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6380..6484)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6552..6570
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6608..6630)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6730..6749
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6963..6982
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        7272..7290
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     7272..7289
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 ttctagctgc gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcgtcgggcg
       61 acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc
      121 atcactaggg gttccttgta gttaatgatt aacccgccat gctacttatc tacgtagcca
      181 tgctctataa cattatggcc ttaggtcact tcatctccat ggggttcttc ttctgatttt
      241 ctagaaaatg agatgggggt gcagagagct tcctcagtga cctgcccagg gtcacatcag
      301 aaatgtcaga gctagaactt gaactcagat tactaatctt aaattccatg ccttgggggc
      361 atgcaagtac gatatacaga aggagtgaac tcattagggc agatgaccaa tgagtttagg
      421 aaagaagagt ccagggcagg gtacatctac accacccgcc cagccctggg tgagtccagc
      481 cacgttcacc tcattatagt tgcctctctc cagtcctacc ttgacgggaa gcacaagcag
      541 aaactgggac aggagcccca ggagaccaaa tcttcatggt ccctctggga ggatgggtgg
      601 ggagagctgt ggcagaggcc tcaggagggg ccctgctgct cagtggtgac agataggggt
      661 gagaaagcag acagagtcat tccgtcagca ttctgggtct gtttggtact tcttctcacg
      721 ctaaggtggc ggtgtgatat gcacaatggc taaaaagcag ggagagctgg aaagaaacaa
      781 ggacagagac agaggccaag tcaaccagac caattcccag aggaagcaaa gaaaccatta
      841 cagagactac aagggggaag ggaaggagag atgaattagc ttcccctgta aaccttagaa
      901 cccagctgtt gccagggcaa cggggcaata cctgtctctt cagaggagat gaagttgcca
      961 gggtaactac atcctgtctt tctcaaggac catcccagaa tgtggcaccc actagccgtt
     1021 accatagcaa ctgcctcttt gccccactta atcccatccc gtctgttaaa agggccctat
     1081 agttggaggt gggggaggta ggaagagcga tgatcacttg tggactaagt ttgttcgcat
     1141 ccccttctcc aaccccctca gtacatcacc ctgggggaac agggtccact tgctcctggg
     1201 cccacacagt cctgcagtat tgtgtatata aggccagggc aaagaggagc aggttttaaa
     1261 gtgaaaggca ggcaggtgtt ggggaggcag ttaccggggc aacgggaaca gggcgtttcg
     1321 gaggtggttg ccatggggac ctggatgctg acgaaggctc gcgaggctgt gagcagccac
     1381 agtgccctgc tcagaagccc caagctcgtc agtcaagccg gttctccgtt tgcactcagg
     1441 agcacgggca ggcgagtggc ccctagttct gggggcagcg ggggatccgt cgacccacca
     1501 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     1561 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     1621 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     1681 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
     1741 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     1801 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     1861 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     1921 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     1981 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     2041 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     2101 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     2161 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagggca
     2221 gtggagaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat cctggcccag
     2281 cggccgcagc tcctaagaag aagaggaagg tgatgagcca gttcgacatc ctgtgcaaga
     2341 ccccccccaa ggtgctggtg cggcagttcg tggagagatt cgagaggccc agcggcgaga
     2401 agatcgccag ctgtgccgcc gagctgacct acctgtgctg gatgatcacc cacaacggca
     2461 ccgccatcaa gagggccacc ttcatgagct acaacaccat catcagcaac agcctgagct
     2521 tcgacatcgt gaacaagagc ctgcagttca agtacaagac ccagaaggcc accatcctgg
     2581 aggccagcct gaagaagctg atccccgcct gggagttcac catcatccct tacaacggcc
     2641 agaagcacca gagcgacatc accgacatcg tgtccagcct gcagctgcag ttcgagagca
     2701 gcgaggaggc cgacaagggc aacagccaca gcaagaagat gctgaaggcc ctgctgtccg
     2761 agggcgagag catctgggag atcaccgaga agatcctgaa cagcttcgag tacaccagca
     2821 ggttcaccaa gaccaagacc ctgtaccagt tcctgttcct ggccacattc atcaactgcg
     2881 gcaggttcag cgacatcaag aacgtggacc ccaagagctt caagctggtg cagaacaagt
     2941 acctgggcgt gatcattcag tgcctggtga ccgagaccaa gacaagcgtg tccaggcaca
     3001 tctacttttt cagcgccaga ggcaggatcg accccctggt gtacctggac gagttcctga
     3061 ggaacagcga gcccgtgctg aagagagtga acaggaccgg caacagcagc agcaacaagc
     3121 aggagtacca gctgctgaag gacaacctgg tgcgcagcta caacaaggcc ctgaagaaga
     3181 acgcccccta ccccatcttc gctatcaaga acggccctaa gagccacatc ggcaggcacc
     3241 tgatgaccag ctttctgagc atgaagggcc tgaccgagct gacaaacgtg gtgggcaact
     3301 ggagcgacaa gagggcctcc gccgtggcca ggaccaccta cacccaccag atcaccgcca
     3361 tccccgacca ctacttcgcc ctggtgtcca ggtactacgc ctacgacccc atcagcaagg
     3421 agatgatcgc cctgaaggac gagaccaacc ccatcgagga gtggcagcac atcgagcagc
     3481 tgaagggcag cgccgagggc agcatcagat accccgcctg gaacggcatc atcagccagg
     3541 aggtgctgga ctacctgagc agctacatca acaggcggat ctgagaattc aagctttaac
     3601 agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     3661 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     3721 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
     3781 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
     3841 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
     3901 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
     3961 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc
     4021 cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
     4081 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
     4141 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
     4201 atcgataccg tcgactcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     4261 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     4321 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     4381 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     4441 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcga ctagagcatg
     4501 gctacgtaga taagtagcat ggcgggttaa tcattaacta caaggaaccc ctagtgatgg
     4561 agttggccac tccctctctg cgcgctcgct cgctcactga ctcgctgcgc tcggtcgttc
     4621 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4681 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4741 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4801 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4861 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4921 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4981 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     5041 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     5101 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5161 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     5221 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5281 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5341 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5401 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5461 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5521 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5581 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5641 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5701 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5761 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5821 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5881 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5941 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     6001 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     6061 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     6121 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6181 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6241 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6301 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6361 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6421 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6481 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     6541 taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg
     6601 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     6661 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc
     6721 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccattcgacg ctctccctta
     6781 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
     6841 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
     6901 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
     6961 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc
     7021 acgatgcgtc cggcgtagag gatctggcta gcgatgaccc tgctgattgg ttcgctgacc
     7081 atttccgggt gcgggacggc gttaccagaa actcagaagg ttcgtccaac caaaccgact
     7141 ctgacggcag tttacgagag agatgatagg gtctgcttca gtaagccaga tgctacacaa
     7201 ttaggcttgt acatattgtc gttagaacgc ggctacaatt aatacataac cttatgtatc
     7261 atacacatac gatttaggtg acactataga atacacggaa ttaa
//
