LOCUS       sequence-50206-e        7300 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses ChETA-EYFP in cells lacking Cre (Cre-Off), for optogenetic
            excitation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7300)
  AUTHORS   Saunders A, Johnson CA, Sabatini BL
  TITLE     Novel recombinant adeno-associated viruses for Cre activated and 
            inactivated transgene expression in neurons.
  JOURNAL   Front Neural Circuits. 2012 Jul 27;6:47. doi: 
            10.3389/fncir.2012.00047. eCollection 2012.
  PUBMED    22866029
REFERENCE   2  (bases 1 to 7300)
  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"; journalName: "Front 
            Neural Circuits."; date: "2012-07-27"; volume: "6:47. doi"; pages: "
            10.3389/fncir.2012.00047. eCollection 2012"
COMMENT     Sequence Label: pAAV-Ef1a-DO-ChETA-EYFP-WPRE-pA
FEATURES             Location/Qualifiers
     source          1..7300
                     /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(1450..1483)
                     /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(1534..1567)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      1589..1598
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1595..2521
                     /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
                     PSMLYLATGHRVQWLRYATWLLTCPVILIRLSNLTGLSNDYSRRTMGLLVSDIGTIVWG
                     ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS
                     WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI
                     RKTTKLNIGGTEIEVETLVEDEAEAGAV"
     regulatory      2531..2540
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2537..2539,2540..2542,2543..3256)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2582..2603)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2843..2862)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3190..3211
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    3285..3318
                     /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    3369..3402
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3427..4015
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3480..3500)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3898..3909)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4047..4523
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4246..4265)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4563..4703
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4563..4692
                     /label=AAV2 ITR
     rep_origin      4778..5233
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4865..4884)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5075..5096
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5250..5269)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5369..5391
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5429..5447)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5515..5619
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5620..5688,5689..6480)
                     /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(5838..5857)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6651..7239
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7140..7159
                     /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 gtaccggatc ctctagagtc
     1441 gactccggaa taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga
     1501 ttgtagctgc tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg
     1561 aagttatggc gcgccttatc tagatttagc caccatggac tatggcggcg ctttgtctgc
     1621 cgtcggacgc gaacttttgt tcgttactaa tcctgtggtg gtgaacgggt ccgtcctggt
     1681 ccctgaggat caatgttact gtgccggatg gattgaatct cgcggcacga acggcgctca
     1741 gaccgcgtca aatgtcctgc agtggcttgc agcaggattc agcattttgc tgctgatgtt
     1801 ctatgcctac caaacctgga aatctacatg cggctgggag gagatctatg tgtgcgccat
     1861 tgaaatggtt aaggtgattc tcgagttctt ttttgagttt aagaatccct ctatgctcta
     1921 ccttgccaca ggacaccggg tgcagtggct gcgctatgca acctggctgc tcacttgtcc
     1981 tgtcatcctt atccgcctga gcaacctcac cggcctgagc aacgactaca gcaggagaac
     2041 catgggactc cttgtctcag acatcgggac tatcgtgtgg ggggctacca gcgccatggc
     2101 aaccggctat gttaaagtca tcttcttttg tcttggattg tgctatggcg cgaacacatt
     2161 ttttcacgcc gccaaagcat atatcgaggg ttatcatact gtgccaaagg gtcggtgccg
     2221 ccaggtcgtg accggcatgg catggctgtt tttcgtgagc tggggtatgt tcccaattct
     2281 cttcattttg gggcccgaag gttttggcgt cctgagcgtc tatggctcca ccgtaggtca
     2341 cacgattatt gatctgatga gtaaaaattg ttgggggttg ttgggacact acctgcgcgt
     2401 cctgatccac gagcacatat tgattcacgg agatatccgc aaaaccacca aactgaacat
     2461 cggcggaacg gagatcgagg tcgagactct cgtcgaagac gaagccgagg ccggagccgt
     2521 gccagcggcc gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     2581 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     2641 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     2701 cgtgccctgg cccaccctcg tgaccacctt cggctacggc ctgcagtgct tcgcccgcta
     2761 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     2821 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     2881 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     2941 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     3001 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     3061 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     3121 gctgcccgac aaccactacc tgagctacca gtccgccctg agcaaagacc ccaacgagaa
     3181 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     3241 cgagctgtac aagtaaggtg gcggtggcga ccggtagcgc tagcataact tcgtataaag
     3301 tatcctatac gaagttattt gccttaaccc agaaattatc actgttattc tttagaatgg
     3361 tgcaaagaat aacttcgtat aatgtatgct atacgaagtt atgaattcga tatcaagctt
     3421 atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     3481 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     3541 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     3601 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     3661 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     3721 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     3781 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg
     3841 ctgctcgcct atgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     3901 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     3961 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga
     4021 taccgagcgc tgctcgagag atctacgggt ggcatccctg tgacccctcc ccagtgcctc
     4081 tcctggccct ggaagttgcc actccagtgc ccaccagcct tgtcctaata aaattaagtt
     4141 gcatcatttt gtctgactag gtgtccttct ataatattat ggggtggagg ggggtggtat
     4201 ggagcaaggg gcaagttggg aagacaacct gtagggcctg cggggtctat tgggaaccaa
     4261 gctggagtgc agtggcacaa tcttggctca ctgcaatctc cgcctcctgg gttcaagcga
     4321 ttctcctgcc tcagcctccc gagttgttgg gattccaggc atgcatgacc aggctcagct
     4381 aatttttgtt tttttggtag agacggggtt tcaccatatt ggccaggctg gtctccaact
     4441 cctaatctca ggtgatctac ccaccttggc ctcccaaatt gctgggatta caggcgtgaa
     4501 ccactgctcc cttccctgtc cttctgattt tgtaggtaac cacgtgcgga ccgagcggcc
     4561 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
     4621 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
     4681 gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg
     4741 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     4801 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     4861 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     4921 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     4981 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     5041 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     5101 acactcaacc ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc
     5161 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     5221 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     5281 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     5341 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     5401 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     5461 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     5521 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     5581 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     5641 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     5701 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     5761 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     5821 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     5881 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     5941 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     6001 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     6061 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     6121 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     6181 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     6241 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     6301 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     6361 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     6421 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     6481 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     6541 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     6601 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     6661 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     6721 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     6781 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     6841 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     6901 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     6961 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     7021 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     7081 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     7141 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     7201 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     7261 ttacggttcc tggccttttg ctggcctttt gctcacatgt
//
