LOCUS       sequence-200278-        7276 bp    DNA     circular SYN 14-APR-2025
DEFINITION  AAV expression of eNpHR 3.0 fused to EYFP driven by EF1a promoter 
            for optogenetic inhibition.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7276)
  AUTHORS   Gradinaru V, Zhang F, Ramakrishnan C, Mattis J, Prakash R, Diester 
            I, Goshen I, Thompson KR, Deisseroth K
  TITLE     Molecular and cellular approaches for diversifying and extending 
            optogenetics.
  JOURNAL   Cell. 2010 Apr 2. 141(1):154-65.
  PUBMED    20303157
REFERENCE   2  (bases 1 to 7276)
  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: "Cell. 2010 
            Apr 2. 141(1):154-65."
COMMENT     Sequence Label: pAAV-Ef1a-DIO eNpHR 3.0-EYFP
FEATURES             Location/Qualifiers
     source          1..7276
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        231..1409
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          462..1400
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1357..1377
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1440..1473)
                     /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(1524..1557)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(1593..2303,2304..2306))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1635..1656)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1984..2003
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2243..2264
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    3261..3294
                     /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    3345..3378
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3403..3991
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3456..3476)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3874..3885)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4023..4499
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4222..4241)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4539..4679
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4539..4668
                     /label=AAV2 ITR
     rep_origin      4754..5209
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4841..4860)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5051..5072
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5226..5245)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5345..5367
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5405..5423)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5491..5595
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5596..5664,5665..6456)
                     /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(5814..5833)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6627..7215
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7116..7135
                     /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 gcgtaagctt tgcaaagatg gataaagttt taaacagaga
      181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
      241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
      301 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
      361 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
      421 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
      481 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
      541 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
      601 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
      661 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
      721 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
      781 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
      841 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
      901 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
      961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     1021 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     1081 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     1141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     1201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     1261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     1321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     1381 gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc gactccggaa
     1441 taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga ttgtagctgc
     1501 tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg aagttatggc
     1561 gcgcctcatt acacctcgtt ctcgtagcag aacttgtaca gctcgtccat gccgagagtg
     1621 atcccggcgg cggtcacgaa ctccagcagg accatgtgat cgcgcttctc gttggggtct
     1681 ttgctcaggg cggactggta gctcaggtag tggttgtcgg gcagcagcac ggggccgtcg
     1741 ccgatggggg tgttctgctg gtagtggtcg gcgagctgca cgctgccgtc ctcgatgttg
     1801 tggcggatct tgaagttcac cttgatgccg ttcttctgct tgtcggccat gatatagacg
     1861 ttgtggctgt tgtagttgta ctccagcttg tgccccagga tgttgccgtc ctccttgaag
     1921 tcgatgccct tcagctcgat gcggttcacc agggtgtcgc cctcgaactt cacctcggcg
     1981 cgggtcttgt agttgccgtc gtccttgaag aagatggtgc gctcctggac gtagccttcg
     2041 ggcatggcgg acttgaagaa gtcgtgctgc ttcatgtggt cggggtagcg ggcgaagcac
     2101 tgcaggccgt agccgaaggt ggtcacgagg gtgggccagg gcacgggcag cttgccggtg
     2161 gtgcagatga acttcagggt cagcttgccg taggtggcat cgccctcgcc ctcgccggac
     2221 acgctgaact tgtggccgtt tacgtcgccg tccagctcga ccaggatggg caccaccccg
     2281 gtgaacagct cctcgccctt gctcaccacg ttgatgtcga tctggtccag ggggatgtac
     2341 tcgccctcgc tggtgatcct gctcttggcg gccgcatcat cagccggggt cccagaagca
     2401 gatggaacat ccaaaatgct gccggacacg acagattcgt ttgacgtcag atagttcagg
     2461 agcaagaatg cgaaaatgta ctttgccacg atatccagga aagaatatcc ccagctcgtc
     2521 accccaacgg gcaacaccgc gataccctcg actccaagag cccacacaat tggatacccc
     2581 agccacatca ctactgtcaa cagcttcagg gtattgaaca tgtcagcggt tcccgcggct
     2641 ttggcgtcct gtgcccactc caccagcagg atatacaaga ccaccagaaa gcatgcacaa
     2701 ctgatagcgt accagaacca tctcatgagg tgggaggagg tagtcagggc cgcggcaagg
     2761 ccagtcacgc acatagcgat atcgaaagtg atagctgtga agagctttgt agcattgctt
     2821 ccggccagga gacccagagc gaggagaatc atgggcgtgg aaagtgccca ggtgagatac
     2881 cgtccccaca tggtgactac accatctacc tcttctcctc ccagcatcac tgagctgcct
     2941 tctgcaaaat ggcccgctgg catttcaaga acggaaattg tcaggccgct cgccaatcca
     3001 gtgtaggagg caatgctgac gacaggcaca aggatggttg acacagcaat aagttttgcc
     3061 cgtggatcat cgagtcctcg ggtcataaaa acgaacagca gtatactcag tcctgcaagt
     3121 gcgatgttga tatagagact gcttgcaagc aaagggtcgt tcagcacgaa ctcgaacaac
     3181 tccctttggg taacctcggc ttgaagggcc acggcactct cggtcacggg aggcagggtc
     3241 tctgtcatgg tggcgctagc ataacttcgt ataaagtatc ctatacgaag ttatttgcct
     3301 taacccagaa attatcactg ttattcttta gaatggtgca aagaataact tcgtataatg
     3361 tatgctatac gaagttatga attcgatatc aagcttatcg ataatcaacc tctggattac
     3421 aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga
     3481 tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc
     3541 tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa
     3601 cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc
     3661 acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc
     3721 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc
     3781 gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctatgt tgccacctgg
     3841 attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct
     3901 tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg
     3961 agtcggatct ccctttgggc cgcctccccg catcgatacc gagcgctgct cgagagatct
     4021 acgggtggca tccctgtgac ccctccccag tgcctctcct ggccctggaa gttgccactc
     4081 cagtgcccac cagccttgtc ctaataaaat taagttgcat cattttgtct gactaggtgt
     4141 ccttctataa tattatgggg tggagggggg tggtatggag caaggggcaa gttgggaaga
     4201 caacctgtag ggcctgcggg gtctattggg aaccaagctg gagtgcagtg gcacaatctt
     4261 ggctcactgc aatctccgcc tcctgggttc aagcgattct cctgcctcag cctcccgagt
     4321 tgttgggatt ccaggcatgc atgaccaggc tcagctaatt tttgtttttt tggtagagac
     4381 ggggtttcac catattggcc aggctggtct ccaactccta atctcaggtg atctacccac
     4441 cttggcctcc caaattgctg ggattacagg cgtgaaccac tgctcccttc cctgtccttc
     4501 tgattttgta ggtaaccacg tgcggaccga gcggccgcag gaacccctag tgatggagtt
     4561 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg
     4621 acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg
     4681 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca
     4741 aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     4801 cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct
     4861 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     4921 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt
     4981 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     5041 ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat
     5101 tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt
     5161 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact
     5221 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     5281 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     5341 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     5401 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     5461 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     5521 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     5581 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     5641 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     5701 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     5761 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     5821 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     5881 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     5941 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     6001 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     6061 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     6121 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     6181 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     6241 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     6301 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     6361 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     6421 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     6481 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     6541 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     6601 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     6661 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     6721 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     6781 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     6841 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     6901 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     6961 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     7021 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     7081 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     7141 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     7201 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     7261 ccttttgctc acatgt
//
