LOCUS       sequence-67967-e        6588 bp    DNA     circular SYN 14-APR-2025
DEFINITION  An AAV vector that expresses 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 6588)
  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 6588)
  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: "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: pOTTC374 - pAAV EF1a DIO iRFP
FEATURES             Location/Qualifiers
     source          1..6588
                     /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(1588..2538)
                     /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"
     regulatory      2535..2544
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2573..2606
                     /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    2657..2690
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2715..3303
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2768..2788)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3186..3197)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3335..3811
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3534..3553)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3851..3991
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3851..3980
                     /label=AAV2 ITR
     rep_origin      4066..4521
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4153..4172)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4363..4384
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4538..4557)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4657..4679
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4717..4735)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4803..4907
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4908..4976,4977..5768)
                     /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(5126..5145)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5939..6527
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6428..6447
                     /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 gcgccggtac cgtcgactca ctcttccatc acgccgatct gccaggccag
     1621 aacctgggcg actagctcgc aggcttggcg gccatcgaga tcgacgtagt acggcgttcg
     1681 gtgatggcaa acgatcaatc cccacagtcg ctcgccgcgc aaaatcgaga tcgacatcgt
     1741 gccgtgcatg cctatgttgc gcatgaattc cagatggacg ggcgagacgc tgcgcaggat
     1801 ggcgaagcta agatcaatcg gccgcccggt gaccggattg aggtctgggg tgaccggcac
     1861 cggccgataa ttgatatcgg gaatgatccg taccgggttg atggtataga gccgacgggc
     1921 ctgcgccggc acggttgagg caggatagtg caggcctagt tttgactcga cctcggcgca
     1981 ccgatcctct gcgatcactt cgccgctgaa gtcggaggcg aagcgataga tcatcacccg
     2041 atcgaagccg gtaatcttcc gcacctcttg cgccgcggcg gcgcaggcgc tttccaaggt
     2101 ttcggcggcc tgcaggcggc ggatggcgct gttggtgcgg cggaagaacg cctgcggctc
     2161 ggcgacgtcc cgctggggag gctcgagctc gaggaagatg agctgatcat ggcgatgcca
     2221 ggagccgatg aagcctgcgt cctttcgcat cgtgaagccg acagtgatcg gtgctccgac
     2281 ggccgccccg ggctcggcca aggcgatcgt cagacggttg tgcgtctccg agtcgaagac
     2341 atcggccgca gagcggccga tcagggcgcc gatcgccagt ccggtgagtt cgggaaggtt
     2401 gtcgctgccg gcaacgatcg tcatgtcggc ggcgagggcg agcagcagtc catgcggttg
     2461 gatggcaccg gggatatgga tcggctcatc gtcgcaggtc aagaggtcag gctgcctggc
     2521 gacggatcct tcagccatgg tggcggcaga tctgagtccg gtagcgctag cgataacttc
     2581 gtataaagta tcctatacga agttatttgc cttaacccag aaattatcac tgttattctt
     2641 tagaatggtg caaagaataa cttcgtataa tgtatgctat acgaagttat gaattcgata
     2701 tcaagcttat cgataatcaa cctctggatt acaaaatttg tgaaagattg actggtattc
     2761 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg
     2821 ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc
     2881 tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg
     2941 acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg
     3001 ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga
     3061 caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct
     3121 ttccttggct gctcgcctat gttgccacct ggattctgcg cgggacgtcc ttctgctacg
     3181 tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc
     3241 ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc
     3301 cgcatcgata ccgagcgctg ctcgagagat ctacgggtgg catccctgtg acccctcccc
     3361 agtgcctctc ctggccctgg aagttgccac tccagtgccc accagccttg tcctaataaa
     3421 attaagttgc atcattttgt ctgactaggt gtccttctat aatattatgg ggtggagggg
     3481 ggtggtatgg agcaaggggc aagttgggaa gacaacctgt agggcctgcg gggtctattg
     3541 ggaaccaagc tggagtgcag tggcacaatc ttggctcact gcaatctccg cctcctgggt
     3601 tcaagcgatt ctcctgcctc agcctcccga gttgttggga ttccaggcat gcatgaccag
     3661 gctcagctaa tttttgtttt tttggtagag acggggtttc accatattgg ccaggctggt
     3721 ctccaactcc taatctcagg tgatctaccc accttggcct cccaaattgc tgggattaca
     3781 ggcgtgaacc actgctccct tccctgtcct tctgattttg taggtaacca cgtgcggacc
     3841 gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg
     3901 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca
     3961 gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg
     4021 catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg
     4081 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     4141 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     4201 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     4261 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga
     4321 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     4381 ctggaacaac actcaaccct atctcgggct attcttttga tttataaggg attttgccga
     4441 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     4501 aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat
     4561 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     4621 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     4681 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     4741 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     4801 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     4861 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     4921 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     4981 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     5041 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     5101 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     5161 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     5221 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     5281 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     5341 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     5401 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     5461 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     5521 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     5581 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     5641 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     5701 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     5761 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     5821 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     5881 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     5941 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     6001 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     6061 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     6121 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     6181 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     6241 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     6301 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     6361 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     6421 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     6481 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     6541 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt
//
