LOCUS       sequence-398291-        5691 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses optimized PdCO in frame with EGFP under control of minimal
            CamKIIa promotor..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5691)
  AUTHORS   Wietek J, Nozownik A, Pulin M, Saraf-Sinik I, Matosevich N, 
            Gowrishankar R, Gat A, Malan D, Brown BJ, Dine J, Imambocus BN, Levy
            R, Sauter K, Litvin A, Regev N, Subramaniam S, Abrera K, Summarli D,
            Goren EM, Mizrachi G, Bitton E, Benjamin A, Copits BA, Sasse P, Rost
            BR, Schmitz D, Bruchas MR, Soba P, Oren-Suissa M, Nir Y, Wiegert JS,
            Yizhar O
  TITLE     A bistable inhibitory optoGPCR for multiplexed optogenetic control 
            of neural circuits.
  JOURNAL   Nat Methods. 2024 May 29. doi: 10.1038/s41592-024-02285-8.
  PUBMED    38811857
REFERENCE   2  (bases 1 to 5691)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods. 2024 May 29. doi: 10.1038/s41592-024-02285-8."
COMMENT     Sequence Label: pAAV_CaMKIIa(0.4)-PdCO-EGFP-WPRE
FEATURES             Location/Qualifiers
     source          1..5691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     32..51
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   193..322
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     regulatory      720..729
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1902..1904,1905..1907,1908..2618)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1902..1904,1905..1907,1908..2618)
                     /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(1947..1968)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2208..2227)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2555..2576
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2658..3061
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     polyA_signal    3068..3116
                     /note="synthetic polyadenylation signal"
     repeat_region   3146..3286
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3146..3275
                     /label=AAV2 ITR
     rep_origin      3361..3816
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3448..3467)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3658..3679
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3833..3852)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3952..3974
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4012..4030)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4098..4202
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4203..4271,4272..5063)
                     /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(4421..4440)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5234..5691,1..131)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
       61 tcgggttttg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
      121 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
      181 ttgctcacat gtcctgcagg cagctgcgcg ctcgctcgct cactgaggcc gcccgggcgt
      241 cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc
      301 aactccatca ctaggggttc ctgcggccgc acgcgtcttg tggactaagt ttgttcgcat
      361 ccccttctcc aaccccctca gtacatcacc ctgggggaac agggtccact tgctcctggg
      421 cccacacagt cctgcagtat tgtgtatata aggccagggc aaagaggagc aggttttaaa
      481 gtgaaaggca ggcaggtgtt ggggaggcag ttaccggggc aacgggaaca gggcgtttcg
      541 gaggtggttg ccatggggac ctggatgctg acgaaggctc gcgaggctgt gagcagccac
      601 agtgccctgc tcagaagccc caagctcgtc agtcaagccg gttctccgtt tgcactcagg
      661 agcacgggca ggcgagtggc ccctagttct gggggcagct ctagagcggt accggatccg
      721 ccaccatgga cggggagaac ttgacgattc caaatcctgt caccgagttg atggataccc
      781 caatcaacag cacgtatttc caaaacctta atgcggaaac ggacggcgga aatcattaca
      841 tctacaatgc cttcacagcc accgattaca acatttgtgc agcttacctt ttcttcattg
      901 cttgtctggg agtcagtctc aacgttttgg tgttggtcct ctttatcaaa gacagaaagt
      961 tgaggagccc caataacttc ctgtatgtaa gtctcgctct cggggatctg ttggttgcag
     1021 ttttcggcac cgcatttaag ttcattatta cagctcgcaa aaccctgttg cgagaagaag
     1081 acggtttctg caaatggtac ggcttcataa catacctcgg cgggctcgcg gcactgatga
     1141 ccctgagcgt tattgctttt gttcgttgtc tggccgtgct gcgtctcggc tccttcacag
     1201 gtttgacaac gcggatgggc gtggcggcga tggctttcat ttggatctac tccctggcat
     1261 tcacactcgc gcctctcctt gggtggaatc actacatacc ggaaggtttg gccacttggt
     1321 gtagtatcga ctggctttcc gatgaaacat ccgataagtc ctacgtgttt gctattttca
     1381 tcttctgctt tcttgtccct gttctcatta tcgtcgtgtc atacgggctg atctacgata
     1441 aggttcggaa ggtagcgaaa acaggcggtt ctgttgccaa agcagaacgc gaagtactgc
     1501 gcatgacgct gctgatggtt agcctgttca tgcttgcctg gagtccctac gctgttatct
     1561 gcatgctcgc tagcttcggc cctaaagacc tgctccatcc agtggccaca gtgattcctg
     1621 ccatgtttgc aaagtcttct acgatgtata accctcttat ctatgtgttc atgaacaaac
     1681 aattcaggcg gagccttaaa gttcttctcg gaatgggagt agaggacctg aactccgaga
     1741 gtgagagagc aactggtggt acagccacga atcaagtcgc cgcgacgacc gagacttccc
     1801 aggttgctcc tgctccgcga gcccgagatc caaccggtaa gagcaggatc accagcgagg
     1861 gcgagtacat ccccctggac cagatcgaca tcaacgtggt gatggtgagc aagggcgagg
     1921 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     1981 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     2041 tcatttgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     2101 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     2161 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     2221 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     2281 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     2341 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     2401 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     2461 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     2521 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     2581 ccgccgggat cactctcggc atggacgagc tgtacaagtt ctgctacgag aacgaggtgt
     2641 aactagaatt caagcttaat caacctctgg attacaaaat ttgtgaaaga ttgactgata
     2701 ttcttaacta tgttgctcct tttacgctgt gtggatatgc tgctttaatg cctctgtatc
     2761 atgctattgc ttcccgtatg gctttcgttt tctcctcctt gtataaatcc tggttgctgt
     2821 ctctttatga ggagttgtgg cccgttgtcc gtcaacgtgg cgtggtgtgc tctgtgtttg
     2881 ctgacgcaac ccccactggc tggggcattg ccaccacctg tcaactcctt tctgggactt
     2941 tcgctttccc cctcccgatc gccacggcag aactcatcgc cgcctgcctt gcccgctgct
     3001 ggacaggggc taggttgctg ggcactgata attccgtggt gttgtcgggg aagctgacgt
     3061 cagcgctaat aaaatcgcta tccatcgaag atggatgtgt gttggttttt tgtgtgggta
     3121 acccacgtgc ggaccgagcg gccgcaggaa cccctagtga tggagttggc cactccctct
     3181 ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt
     3241 gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg
     3301 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt
     3361 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     3421 ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     3481 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     3541 gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc
     3601 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     3661 gactcttgtt ccaaactgga acaacactca actctatctc gggctattct tttgatttat
     3721 aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta
     3781 acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct
     3841 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     3901 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     3961 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     4021 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     4081 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     4141 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     4201 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     4261 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     4321 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     4381 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     4441 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     4501 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     4561 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     4621 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     4681 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     4741 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     4801 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     4861 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     4921 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     4981 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     5041 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     5101 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     5161 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     5221 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     5281 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     5341 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     5401 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     5461 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     5521 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     5581 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     5641 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc g
//
