LOCUS       sequence-76270-e        7071 bp    DNA     circular SYN 14-APR-2025
DEFINITION  An AAV packaging vector that expresses channel rhodopsin 2 (H134R) 
            (fused to EYFP) under the EF1a promoter, which can be deactivated 
            (deleted) by recombination between the flanking loxP sites..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7071)
  AUTHORS   McDevitt RA, Tiran-Cappello A, Shen H, Balderas I, Britt JP, Marino 
            RAM, Chung SL, Richie CT, Harvey BK, Bonci A
  TITLE     Serotonergic versus nonserotonergic dorsal raphe projection neurons:
            differential participation in reward circuitry.
  JOURNAL   Cell Rep. 2014 Sep 25;8(6):1857-1869. doi: 
            10.1016/j.celrep.2014.08.037. Epub 2014 Sep 18.
  PUBMED    25242321
REFERENCE   2  (bases 1 to 7071)
  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"; doi: 
            "10.1016/j.celrep.2014.08.037"; journalName: "Cell Rep"; date: 
            "2014-09-25- 25"; volume: "8"; issue: "6"; pages: "1857-1869"
COMMENT     Sequence Label: pOTTC292 - pAAV EF1a floxed hChR2(H134R)-EYFP
FEATURES             Location/Qualifiers
     source          1..7071
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        242..1419
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          473..1410
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1367..1387
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1426..1459)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      1464..1473
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1470..2396
                     /codon_start=1
                     /product="H134R mutant of an N-terminal fragment of 
                     channelrhodopsin-2 from the alga Chlamydomonas reinhardtii 
                     (Nagel et al., 2005)"
                     /label=ChR2(H134R)
                     /note="blue light-activated cation channel"
                     /translation="MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGT
                     NGAQTASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKN
                     PSMLYLATGHRVQWLRYAEWLLTCPVILIRLSNLTGLSNDYSRRTMGLLVSDIGTIVWG
                     ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS
                     WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI
                     RKTTKLNIGGTEIEVETLVEDEAEAGAV"
     regulatory      2406..2415
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2412..2414,2415..2417,2418..3131)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2457..2478)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2718..2737)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3065..3086
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(3140..3173)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3198..3786
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3251..3271)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3669..3680)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3818..4294
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4017..4036)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4334..4474
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4334..4463
                     /label=AAV2 ITR
     rep_origin      4549..5004
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4636..4655)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4846..4867
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5021..5040)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5140..5162
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5200..5218)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5286..5390
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5391..5459,5460..6251)
                     /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(5609..5628)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6422..7010
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6911..6930
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtaagct ttgcaaagat ggataaagtt
      181 ttaaacagag aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat
      241 tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg
      301 gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag
      361 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc
      421 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc
      481 cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat
      541 tacttccact ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
      601 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
      661 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
      721 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
      781 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
      841 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
      901 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
      961 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
     1021 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
     1081 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
     1141 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
     1201 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     1261 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     1321 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     1381 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gtaccataac ttcgtatagc
     1441 atacattata cgaagttatg ctagccacca tggactatgg cggcgctttg tctgccgtcg
     1501 gacgcgaact tttgttcgtt actaatcctg tggtggtgaa cgggtccgtc ctggtccctg
     1561 aggatcaatg ttactgtgcc ggatggattg aatctcgcgg cacgaacggc gctcagaccg
     1621 cgtcaaatgt cctgcagtgg cttgcagcag gattcagcat tttgctgctg atgttctatg
     1681 cctaccaaac ctggaaatct acatgcggct gggaggagat ctatgtgtgc gccattgaaa
     1741 tggttaaggt gattctcgag ttcttttttg agtttaagaa tccctctatg ctctaccttg
     1801 ccacaggaca ccgggtgcag tggctgcgct atgcagagtg gctgctcact tgtcctgtca
     1861 tccttatccg cctgagcaac ctcaccggcc tgagcaacga ctacagcagg agaaccatgg
     1921 gactccttgt ctcagacatc gggactatcg tgtggggggc taccagcgcc atggcaaccg
     1981 gctatgttaa agtcatcttc ttttgtcttg gattgtgcta tggcgcgaac acattttttc
     2041 acgccgccaa agcatatatc gagggttatc atactgtgcc aaagggtcgg tgccgccagg
     2101 tcgtgaccgg catggcatgg ctgtttttcg tgagctgggg tatgttccca attctcttca
     2161 ttttggggcc cgaaggtttt ggcgtcctga gcgtctatgg ctccaccgta ggtcacacga
     2221 ttattgatct gatgagtaaa aattgttggg ggttgttggg acactacctg cgcgtcctga
     2281 tccacgagca catattgatt cacggagata tccgcaaaac caccaaactg aacatcggcg
     2341 gaacggagat cgaggtcgag actctcgtcg aagacgaagc cgaggccgga gccgtgccag
     2401 cggccgccac catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     2461 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
     2521 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
     2581 cctggcccac cctcgtgacc accttcggct acggcctgca gtgcttcgcc cgctaccccg
     2641 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     2701 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     2761 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     2821 tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca
     2881 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg
     2941 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     3001 ccgacaacca ctacctgagc taccagtccg ccctgagcaa agaccccaac gagaagcgcg
     3061 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     3121 tgtacaagta aggcgcgcca taacttcgta tagcatacat tatacgaagt tatgaattcg
     3181 atatcaagct tatcgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta
     3241 ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc
     3301 atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt
     3361 ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg
     3421 ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt
     3481 tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct
     3541 ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt
     3601 cctttccttg gctgctcgcc tatgttgcca cctggattct gcgcgggacg tccttctgct
     3661 acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc
     3721 ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct
     3781 ccccgcatcg ataccgagcg ctgctcgaga gatctacggg tggcatccct gtgacccctc
     3841 cccagtgcct ctcctggccc tggaagttgc cactccagtg cccaccagcc ttgtcctaat
     3901 aaaattaagt tgcatcattt tgtctgacta ggtgtccttc tataatatta tggggtggag
     3961 gggggtggta tggagcaagg ggcaagttgg gaagacaacc tgtagggcct gcggggtcta
     4021 ttgggaacca agctggagtg cagtggcaca atcttggctc actgcaatct ccgcctcctg
     4081 ggttcaagcg attctcctgc ctcagcctcc cgagttgttg ggattccagg catgcatgac
     4141 caggctcagc taatttttgt ttttttggta gagacggggt ttcaccatat tggccaggct
     4201 ggtctccaac tcctaatctc aggtgatcta cccaccttgg cctcccaaat tgctgggatt
     4261 acaggcgtga accactgctc ccttccctgt ccttctgatt ttgtaggtaa ccacgtgcgg
     4321 accgagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc
     4381 tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc
     4441 tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt
     4501 acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta
     4561 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     4621 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     4681 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     4741 acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat
     4801 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     4861 aaactggaac aacactcaac cctatctcgg gctattcttt tgatttataa gggattttgc
     4921 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     4981 acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg
     5041 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     5101 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     5161 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     5221 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     5281 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     5341 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     5401 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     5461 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     5521 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     5581 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     5641 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     5701 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     5761 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     5821 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     5881 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     5941 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     6001 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     6061 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     6121 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     6181 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     6241 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     6301 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     6361 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     6421 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     6481 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     6541 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     6601 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     6661 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     6721 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     6781 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     6841 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     6901 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     6961 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     7021 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg t
//
