LOCUS       sequence-465970-        7835 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bicistronic Cre-dependent expression of TRPV4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7835)
  AUTHORS   Wheeler MA, Smith CJ, Ottolini M, Barker BS, Purohit AM, Grippo RM, 
            Gaykema RP, Spano AJ, Beenhakker MP, Kucenas S, Patel MK, Deppmann 
            CD, Guler AD
  TITLE     Genetically targeted magnetic control of the nervous system.
  JOURNAL   Nat Neurosci. 2016 Mar 7. doi: 10.1038/nn.4265.
  PUBMED    26950006
REFERENCE   2  (bases 1 to 7835)
  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: "Nat 
            Neurosci. 2016 Mar 7. doi: 10.1038/nn.4265."
COMMENT     Sequence Label: pAAV-CMV-DIO-TRPV4-p2A-ferritin-sNRPpA
FEATURES             Location/Qualifiers
     source          1..7835
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        196..499
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        500..703
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     654..674
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    complement(766..799)
                     /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(850..883)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(931..954)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(1549..1572)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(2143..2166)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(2200..2256)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             complement(2272..2295)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      4905..4914
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    4927..4960
                     /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    5011..5044
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     repeat_region   5098..5238
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5098..5227
                     /label=AAV2 ITR
     rep_origin      5313..5768
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5400..5419)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5610..5631
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5785..5804)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5904..5926
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5964..5982)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6050..6154
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6155..6223,6224..7015)
                     /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(6373..6392)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7186..7774
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7675..7694
                     /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 gcggccgcac gcgcctagta atcaattacg gggtcattag ttcatagccc
      181 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
      241 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      301 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
      361 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      421 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
      481 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
      541 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
      601 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
      661 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga
      721 acccactgct tactggctta tcgaaattct agagtcgact ccggaataac ttcgtatagg
      781 atactttata cgaagttatg cagaatggta gctggattgt agctgctatt agcaatatga
      841 aacctcttaa taacttcgta tagcatacat tatacgaagt tatggcgcgc cggatcctta
      901 gctagccact cgggaggagc ctccaccccc tttgtcatcg tcgtctttat agtcgctccc
      961 gccacccccg cgtgcgcttt cattatcact gtctcccagg gtgtgcttgt caaagagata
     1021 ttccgccaag ccagattcgg gcgctcccat cttgcgcaag ttggtcacgt ggtcacccaa
     1081 ttctttgatg gctttcacct gctcattcag gtaatgtgtc tcaatgaagt cacacaaatg
     1141 ggggtcattt ttgtcagtgg ccagtttgtg cagttccagt agtgactgat tcacattttt
     1201 ttccaaatgt aatgcacact ccattgcatt cagcccgctc tcccagtcat cacagtctgg
     1261 tttcttgata tcctgaagga agattcggcc acctcgttgg ttctgcagct tcatcagttt
     1321 ctcagcatgt tccctctcct catgagattg gtgaagaaag tatttggcaa agttcttcaa
     1381 agccacatca tcgcggtcaa agtagtaaga catggacagg taaacgtagg aggcgtagag
     1441 ctccaggttg atctggcggt tgatggcggc ctctgagtcc tggtggtagt tctggcgcac
     1501 ctgcgaggtg gacgcggtcg tcatgacgcg ggaggagccg ccgccgcctt tatcatcatc
     1561 atctttataa tcggagccgc cgccgccgcg ggcgtcgtgc ttgagagtga gcctttcgaa
     1621 gagatactcg cccagcccag cctccgggcc acccagcctg tggaggttgg tcaggtggtc
     1681 acccatcttc ttgataagct tcacttcctc atctaggaag tgagtctcca ggaagtcaca
     1741 gagatggggg tccgtgcggg cagaacccag ggcatgaaga tccaaaaggg cctggttcag
     1801 ctttttctcc agggccatgg cagctttcat ggcgtctggg gttttacccc actcatcttc
     1861 agctggcttc ttgatgtcct ggaagagagc gcggccgcca cgctggtttt gcatcttcag
     1921 gagacgctcg tagccctcgc gcttctcctc ggccaattcg cggaagaagt ggctcacgcc
     1981 ttccagagcc acatcatcgc ggtcgaaata gaagcccaga gagaggtagg tgtaggaggc
     2041 ctgcaggtac aaattgacca ggctgttgac ggctgcctcc acgtcggtgg aataattctg
     2101 acgaatctgg gagcccataa cccgagagga gccgcctcct cctttgtcgt cgtcgtcctt
     2161 ataatcagaa ccgcccccgc cccgtgctcc ggactcgagg gggccggggt tctcctccac
     2221 gtcgccggcc tgcttcagca ggctgaagtt ggtggcgccg ctgccggatc ccttatcgtc
     2281 atcgtccttg taatccagtg gtgcgtcctc cgccctccac ttgggagcat aaccttgctg
     2341 gtggccgtca cagttggggt tccctaggtt atccaggggg accaccactt catctgtgcc
     2401 tgagttcttg ttcagctcca ccacgcgggg caccactgag gaccagcgat ccctgcggag
     2461 gcgccccatg gtatgggaga agccatagta ctggtagatc tcgctcttgc cggggtcctc
     2521 gttaatgatg cccaggttct ggttccagtg agaccagttc acctcgtcca ccctgaagca
     2581 ccacctgcgg tctggagtgc catccgagct cttgcccact gtcaccatct ctccggagcg
     2641 gaaggccttc ctcaggaaca cagggaagga gcgctcgatg tccaggatgg tggtggccca
     2701 ctgcagcttc cagatgtgct tgctctcctt ggacacctgg cccacggtct cacccatgag
     2761 ggcgatgagc atgttcagca ggagcacgaa ggtgaggatg atgtaggtaa ccagcaggag
     2821 aatgaagacc acggggtact tagcgctgct cagcatctcc aggtcgccca tgccgatggt
     2881 gagcttgaag aggtccagta ggaaggcgct gaaggtctcg ctgtcccggc acgcggggta
     2941 tgagggcacc gtgcagttgc tctggtcctc gttacagacc ttcatgttgg tgcacggatt
     3001 caggagggtg accagagctg aggcatagcc aatcataaaa agcaggtaga ccagcagaaa
     3061 gcggaagaga tctttgaaga ggatcttctg aatcatgatg ctgtaggtcc ctgtcagctt
     3121 cagcccacgg gtgaagtaaa gggcattcat ccagcccagg accagggcaa agaccatcac
     3181 agccagatag gcctcgatcc ctgccaggta gagcgccgca gacacaacca ccagcactga
     3241 gtagatgaag tagagcaact ggaaggagcc atcgacgaag agagaattca ctccagggca
     3301 tttcttcatg aacaagtctt tgatactggt aaagaagaac aggactcctg tgaggagcgt
     3361 gatgacctca ccagccagcc tcaggtagtc caccgtggta cggtaagggt agggtggcgt
     3421 gccctccagt ggctgatagt aggctgtgag ggtgaagatg accatggcac acagatagga
     3481 gacaacgttg atgtagaagg acacggcccc gaacttacgc cacttgtccc tcagcagttc
     3541 gttaatgggc tccacagcca gcatctcatg gcggttctcg atcttgctgt tgtaaaccag
     3601 gatctccagc acggacactt cctccccgca cgtatccagg gaggagaggt cgtagagaga
     3661 agaatacaca ggcccgtagg cccagtcctt gaacttgcga gacaggtgcc gtgtgtcctc
     3721 atctgtcacc tcccgtcgga tgatgtgctg aaagaccccg atcttgccag tcttggcagc
     3781 catcatgagg ggcgaaagac cgtcattgtt aagcacagtc tccaggttgc tgtctgggaa
     3841 gaggcgggag cacttgagaa gcaacaggtc atacatcttg gtgacaaact tggtgttctc
     3901 tcgggtgttg tcagcgatgg ccaccagcgc gtggagcacc gtgttgcctc tggagtcctg
     3961 tcgcctcata tcggctttct tgtgagggtt ctctgtcagg tagttgacga tgtgcggctg
     4021 gttggtgcag gctgccaagg acaagggcag ctccccaaag tagaagtagc caccctcatc
     4081 cttgggctgg aagaaccgcc ctcgggcctg cgcgtgcaca tcggctccct gggccaccag
     4141 gagctccacg taatgcttgc agcgccgttc aatggcgatg tgcagtgccg tctgccctcg
     4201 gtagtagatg tctctgaagg gcgagttgat gaactcccgc atgttgcccg tgcgttccgc
     4261 aatgtccagc aacactggga tggtgtcgtt tcggccattg cttaagttca gaagtgcctt
     4321 gggcaggcag gtcttccctg tggatggttc ccggaactcc tcatcagtca ggcgcttctt
     4381 gtgggtcagc aagtaggaga gcagtccgtc caggtcggca gtggagcccc gggacacgat
     4441 gtcaaagagg atgggccggt tgaagacttt gaggatgggg ggtggctggg gggcgggagc
     4501 tttggggctc tgtggctgct tctctacgac cttcctcctc catctcttgt tgtcactggg
     4561 gtggtgccgg taagtgccat agtcgaacaa cgaatccatg ggcgctttct tgggcccagg
     4621 cactactgag gactcataca gggtggactc cagcaggtca atggggttgg gaaccccctt
     4681 gcggaaagcg ccctggaact tcatacgcag gtttggacgt ccatccccgg ggccagcagg
     4741 gcggctagca tccactggtg aaagagaaga ggagccttcc tctccctcaa acaggttggc
     4801 cagggaagag agggggaagg cctccccacc agaagtgcca ctctcgtctc cagggggctc
     4861 agccacatcc ccaggcgctg cacgggggcc atcaccagga tctgccatgg tggcggtacc
     4921 tctagcataa cttcgtataa agtatcctat acgaagttat ttgccttaac ccagaaatta
     4981 tcactgttat tctttagaat ggtgcaaaga ataacttcgt ataatgtatg ctatacgaag
     5041 ttatgaattc aaataaaata cgaaatgcac gtaaccacgt gcggaccgag cggccgcagg
     5101 aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg
     5161 ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag
     5221 cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     5281 tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc
     5341 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc
     5401 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     5461 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     5521 acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     5581 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     5641 caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg
     5701 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt
     5761 tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     5821 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     5881 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     5941 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     6001 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     6061 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     6121 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     6181 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     6241 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     6301 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     6361 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     6421 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     6481 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     6541 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     6601 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     6661 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     6721 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     6781 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     6841 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     6901 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     6961 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     7021 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     7081 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     7141 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     7201 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     7261 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     7321 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     7381 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     7441 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     7501 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     7561 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     7621 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     7681 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     7741 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     7801 gttcctggcc ttttgctggc cttttgctca catgt
//
