LOCUS       sequence-68005-e        7564 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An AAV vector that expresses channel rhodopsin 2 (H134R) (fused to 
            iRFP) in a Cre-dependent manner (DIO, double floxed inverted open 
            reading frame)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7564)
  AUTHORS   Richie CT, Whitaker LR, Whitaker KW, Necarsulmer J, Baldwin HA, 
            Zhang Y, Fortuno L, Hinkle JJ, Koivula P, Henderson MJ, Sun W, Wang 
            K, Smith JC, Pickel J, Ji N, Hope BT, Harvey BK
  TITLE     Near-infrared fluorescent protein iRFP713 as a reporter protein for 
            optogenetic vectors, a transgenic Cre-reporter rat, and other 
            neuronal studies.
  JOURNAL   J Neurosci Methods. 2017 Jun 1;284:1-14. doi: 
            10.1016/j.jneumeth.2017.03.020. Epub 2017 Apr 2.
  PUBMED    28380331
REFERENCE   2  (bases 1 to 7564)
  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: "J Neurosci 
            Methods."; date: "2017-06-1"; volume: "284:1-14. doi"; pages: " 
            10.1016/j.jneumeth.2017.03.020. Epub 2017 Apr 2"
COMMENT     Sequence Label: pOTTC468 - pAAV EF1a DIO hChR2(H134R)-iRFP
FEATURES             Location/Qualifiers
     source          1..7564
                     /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)."
     CDS             complement(1602..2552)
                     /codon_start=1
                     /product="phytochrome-based near-infrared fluorescent 
                     protein, originally termed iRFP (Filonov et al., 2011)"
                     /label=iRFP713
                     /note="name was changed to iRFP713 (Shcherbakova and 
                     Verkhusha, 2013)"
                     /translation="MAEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGS
                     DNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFI
                     GSWHRHDQLIFLELEPPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQEVRKIT
                     GFDRVMIYRFASDFSGEVIAEDRCAEVESKLGLHYPASTVPAQARRLYTINPVRIIPDI
                     NYRPVPVTPDLNPVTGRPIDLSFAILRSVSPVHLEFMRNIGMHGTMSISILRGERLWGL
                     IVCHHRTPYYVDLDGRQACELVAQVLAWQIGVMEE"
     CDS             complement(2571..3497)
                     /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      3494..3503
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3549..3582
                     /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    3633..3666
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3691..4279
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3744..3764)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4162..4173)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4311..4787
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4510..4529)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4827..4967
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4827..4956
                     /label=AAV2 ITR
     rep_origin      5042..5497
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5129..5148)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5339..5360
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5514..5533)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5633..5655
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5693..5711)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5779..5883
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5884..5952,5953..6744)
                     /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(6102..6121)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6915..7503
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7404..7423
                     /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 gcgccataag cttgatatcg aattcgtcga ctcactcttc catcacgccg
     1621 atctgccagg ccagaacctg ggcgactagc tcgcaggctt ggcggccatc gagatcgacg
     1681 tagtacggcg ttcggtgatg gcaaacgatc aatccccaca gtcgctcgcc gcgcaaaatc
     1741 gagatcgaca tcgtgccgtg catgcctatg ttgcgcatga attccagatg gacgggcgag
     1801 acgctgcgca ggatggcgaa gctaagatca atcggccgcc cggtgaccgg attgaggtct
     1861 ggggtgaccg gcaccggccg ataattgata tcgggaatga tccgtaccgg gttgatggta
     1921 tagagccgac gggcctgcgc cggcacggtt gaggcaggat agtgcaggcc tagttttgac
     1981 tcgacctcgg cgcaccgatc ctctgcgatc acttcgccgc tgaagtcgga ggcgaagcga
     2041 tagatcatca cccgatcgaa gccggtaatc ttccgcacct cttgcgccgc ggcggcgcag
     2101 gcgctttcca aggtttcggc ggcctgcagg cggcggatgg cgctgttggt gcggcggaag
     2161 aacgcctgcg gctcggcgac gtcccgctgg ggaggctcga gctcgaggaa gatgagctga
     2221 tcatggcgat gccaggagcc gatgaagcct gcgtcctttc gcatcgtgaa gccgacagtg
     2281 atcggtgctc cgacggccgc cccgggctcg gccaaggcga tcgtcagacg gttgtgcgtc
     2341 tccgagtcga agacatcggc cgcagagcgg ccgatcaggg cgccgatcgc cagtccggtg
     2401 agttcgggaa ggttgtcgct gccggcaacg atcgtcatgt cggcggcgag ggcgagcagc
     2461 agtccatgcg gttggatggc accggggata tggatcggct catcgtcgca ggtcaagagg
     2521 tcaggctgcc tggcgacgga tccttcagcc atactagtgg cggccgctgg cacggctccg
     2581 gcctcggctt cgtcttcgac gagagtctcg acctcgatct ccgttccgcc gatgttcagt
     2641 ttggtggttt tgcggatatc tccgtgaatc aatatgtgct cgtggatcag gacgcgcagg
     2701 tagtgtccca acaaccccca acaattttta ctcatcagat caataatcgt gtgacctacg
     2761 gtggagccat agacgctcag gacgccaaaa ccttcgggcc ccaaaatgaa gagaattggg
     2821 aacatacccc agctcacgaa aaacagccat gccatgccgg tcacgacctg gcggcaccga
     2881 ccctttggca cagtatgata accctcgata tatgctttgg cggcgtgaaa aaatgtgttc
     2941 gcgccatagc acaatccaag acaaaagaag atgactttaa catagccggt tgccatggcg
     3001 ctggtagccc cccacacgat agtcccgatg tctgagacaa ggagtcccat ggttctcctg
     3061 ctgtagtcgt tgctcaggcc ggtgaggttg ctcaggcgga taaggatgac aggacaagtg
     3121 agcagccact ctgcatagcg cagccactgc acccggtgtc ctgtggcaag gtagagcata
     3181 gagggattct taaactcaaa aaagaactcg agaatcacct taaccatttc aatggcgcac
     3241 acatagatct cctcccagcc gcatgtagat ttccaggttt ggtaggcata gaacatcagc
     3301 agcaaaatgc tgaatcctgc tgcaagccac tgcaggacat ttgacgcggt ctgagcgccg
     3361 ttcgtgccgc gagattcaat ccatccggca cagtaacatt gatcctcagg gaccaggacg
     3421 gacccgttca ccaccacagg attagtaacg aacaaaagtt cgcgtccgac ggcagacaaa
     3481 gcgccgccat agtccatggt ggctccggag tcgactctag aggatccggt accgctctag
     3541 agctagcgat aacttcgtat aaagtatcct atacgaagtt atttgcctta acccagaaat
     3601 tatcactgtt attctttaga atggtgcaaa gaataacttc gtataatgta tgctatacga
     3661 agttatgaat tcgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa
     3721 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
     3781 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
     3841 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
     3901 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
     3961 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
     4021 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg
     4081 gggaaatcat cgtcctttcc ttggctgctc gcctatgttg ccacctggat tctgcgcggg
     4141 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg
     4201 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc
     4261 ctttgggccg cctccccgca tcgataccga gcgctgctcg agagatctac gggtggcatc
     4321 cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca gtgcccacca
     4381 gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc ttctataata
     4441 ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca acctgtaggg
     4501 cctgcggggt ctattgggaa ccaagctgga gtgcagtggc acaatcttgg ctcactgcaa
     4561 tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg ttgggattcc
     4621 aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagagacgg ggtttcacca
     4681 tattggccag gctggtctcc aactcctaat ctcaggtgat ctacccacct tggcctccca
     4741 aattgctggg attacaggcg tgaaccactg ctcccttccc tgtccttctg attttgtagg
     4801 taaccacgtg cggaccgagc ggccgcagga acccctagtg atggagttgg ccactccctc
     4861 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt
     4921 tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg
     4981 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag
     5041 tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     5101 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     5161 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     5221 agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg
     5281 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     5341 ggactcttgt tccaaactgg aacaacactc aaccctatct cgggctattc ttttgattta
     5401 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     5461 aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc
     5521 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     5581 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     5641 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg
     5701 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     5761 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     5821 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     5881 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     5941 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     6001 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     6061 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     6121 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     6181 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     6241 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     6301 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     6361 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     6421 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     6481 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     6541 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     6601 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     6661 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     6721 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     6781 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     6841 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     6901 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     6961 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     7021 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     7081 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     7141 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     7201 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     7261 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     7321 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     7381 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     7441 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     7501 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     7561 atgt
//
