LOCUS Exported 8669 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Optogenetic tool for blue-light inhibition and red-light excitation of neurons. ACCESSION . VERSION . KEYWORDS Ef1a-DIO-BiPOLES-mCerulean SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8669) AUTHORS Vierock J, Rodriguez-Rozada S, Pieper F, Dieter A, Bergs A, Zeitzschel N, Ahlbeck J, Sauter K, Gottschalk A, Engel AK, Hegemann P, Wiegert JS TITLE BiPOLES: a tool for bidirectional dual-color optogenetic control of neurons JOURNAL bioRxiv (2020) REFERENCE 2 (bases 1 to 8669) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8669 /organism="synthetic DNA construct" /mol_type="other DNA" 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 1440..1473 /label=lox2272 /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GGATACTT). lox2272 sites are compatible with each other, but incompatible with loxP or loxN sites (Lee and Saito, 1988)." protein_bind 1524..1557 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS complement(1569..2615) /codon_start=1 /product="red light-drivable channelrhodopsin from Chlamydomonas noctigama (Klapoetke et al., 2014)" /label=Chrimson /translation="ELISSATRSLFAAGGINPWPNPYHHEDMGCGGMTPTGECFSTEWW CDPSYGLSDAGYGYCFVEATGGYLVVGVEKKQAWLHSRGTPGEKIGAQVCQWIAFSIAI ALLTFYGFSAWKATCGWEEVYVCCVEVLFVTLEIFKEFSSPATVYLSTGNHAYCLRYFE WLLSCPVILIKLSNLSGLKNDYSKRTMGLIVSCVGMIVFGMAAGLATDWLKWLLYIVSC IYGGYMYFQAAKCYVEANHSVPKGHCRMVVKLMAYAYFASWGSYPILWAVGPEGLLKLS PYANSIGHSICDIIAKEFWTFLAHHLRIKIHEHILIHGDIRKTTKMEIGGEEVEVEEFV EEEDEDTV" primer_bind complement(3015..3036) /label=EGFP-C /note="EGFP, forward primer" primer_bind 3364..3383 /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 3623..3644 /label=EGFP-N /note="EGFP, reverse primer" protein_bind complement(4654..4687) /label=lox2272 /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GGATACTT). lox2272 sites are compatible with each other, but incompatible with loxP or loxN sites (Lee and Saito, 1988)." protein_bind complement(4738..4771) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 4796..5384 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(4849..4869) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(5267..5278) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 5416..5892 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(5615..5634) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 5932..6072 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5932..6061 /label=AAV2 ITR rep_origin 6147..6602 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6234..6253) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6444..6465 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6619..6638) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6738..6760 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6798..6816) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6884..6988 /gene="bla" /label=AmpR promoter CDS 6989..7849 /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(7207..7226) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 8020..8608 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8509..8528 /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 gcgagctttc acactgtgtc ctcgtcctcc tcctccacga actcttccac ttccacctcc 1621 tcgcctccga tctccatctt ggtggtcttc cggatgtcgc cgtggatcag gatgtgctcg 1681 tggatcttga tcctcaggtg gtgggccagg aaggtccaaa actccttggc gatgatgtcg 1741 cagatgctgt ggccgatgct gttggcgtaa gggctcagct tcagcagtcc ttctggtccc 1801 actgcccaga ggattgggta gctgccccaa gaggcgaagt aagcgtaggc catcagcttc 1861 acgaccatgc ggcaatggcc tttaggcacg ctgtggttgg cttccacgta gcacttggcg 1921 gcctggaagt acatgtagcc gccgtagatg caagacacga tatacagcag ccacttgagc 1981 caatcggtag ccagtcctgc ggccatgccg aacacgatca ttcccacgca agacacgatc 2041 aggcccatgg tccgcttgct gtagtcgttc ttcaggccgc tcaggttgct cagcttgatc 2101 aggatcacgg ggcaagacag cagccactcg aagtagcgca ggcaataggc gtggttgccg 2161 gtagacaggt acactgtggc ggggctgctg aactccttga agatctccag ggtcacgaac 2221 agcacctcga cgcaacagac gtagacctcc tcccaaccgc aagtggcctt ccaagcgctg 2281 aagccgtaga atgtcagcag ggcgatggcg atgctgaaag caatccactg gcagacctgg 2341 gcgccgatct tttctcctgg tgtgcctctg ctgtgcagcc aagcctgctt cttctccact 2401 cccacgacca ggtagccgcc tgtggcctcc acgaagcaat atccgtatcc ggcgtcgctc 2461 agtccgtaag aagggtcaca ccaccactcg gtgctgaagc actcgcctgt aggtgtcatt 2521 cctccacagc ccatgtcctc gtggtggtag gggttaggcc aagggttgat gcctccggcg 2581 gcaaacagag atctggtggc gctgctgatc agctcttgta cagctccctc cggacgtccg 2641 gtgatctggg cctgtctaga gctgttttca gagatgttgt aggaaatctg cagccctctt 2701 tccccataca catccggtct caaggttacc cccggtgact ttaactggtc ctggtagtcg 2761 ggggtgtagg gatcaatggt ctgcatcagc acatagatgc acaaggcaaa gagcccagtc 2821 atgaccacgt agaaagctgc atagtacagg ctgatccaca cccaccgggc tggggtgcgg 2881 cccagcatct gcccagtgtc cgggttccaa cagtattgcc ggaattcggc catgcgctgg 2941 ctgcatgact tcttctcctg cagggctgca ccggtataca gctcgtccat gccgagagtg 3001 atcccggcgg cggtcacgaa ctccagcagg accatgtgat cgcgcttctc gttggggtct 3061 ttgctcagct tggactgggt gctcaggtag tggttgtcgg gcagcagcac ggggccgtcg 3121 ccgatggggg tgttctgctg gtagtggtcg gcgagctgca cgctgccgtc ctcgatgttg 3181 cagttgaggc cgaagttggc cttgatgccg ttcttctgct tgtcggcggt gatatagacg 3241 ttgccgtgga tggcgttgta ctccagcttg tgccccagga tgttgccgtc ctccttgaag 3301 tcgatgccct tcagctcgat gcggttcacc agggtgtcgc cctcgaactt cacctcggcg 3361 cgggtcttgt agttgccgtc gtccttgaag aagatggtgc gctcctggac gtagccttcg 3421 ggcatggcgg acttgaagaa gtcgtgctgc ttcatgtggt cggggtagcg ggcgaagcac 3481 tgcacgcccc agctcagggt ggtcacgagg gtgggccagg gcacgggcag cttgccggtg 3541 gtgcagatga acttcagggt cagcttgccg taggtggcat cgccctcgcc ctcgccggac 3601 acgctgaact tgtggccgtt tacgtcgccg tccagctcga ccaggatggg caccaccccg 3661 gtgaacagct cctcgccctt gctcacgggt accgccacgt tgatgtcgat ctggtccagg 3721 gggatgtact cgccctcgct ggtgatcctg ctcttggccg ccgtacgtcc ccttccaaac 3781 attctgatat tgacttcgtc gttaggaggg gcattcttct cgtgattcag gccaaaagga 3841 tcgtcggctt cgggtttctc ctgccctggc agacatgcgg ggtcccactt cccttccagc 3901 actccccagg cagtgctcca caggatcact ccatatgtat ttttgcacag gccgtcagcc 3961 agcaggtaca gaatggcgct cacgttctcg tggatcacgc agactccggt gggtgaaatc 4021 agccacacga ttgggaacat agtccaagca ataaagaaga tcaggcgcag catcttgatc 4081 ctcaccacca ccttctgtgc cagaggtgtc ccgacagatt caaagccata cagccccact 4141 tgaaagattg tgaagaccac actgtactcg aacaccaggc aggtggcccc aaagaaaaag 4201 aacagccact tcatatactc tgcgggagaa attgtagcgg tgattcccat gaccactcta 4261 atgatggaag cggcctgtgc aatgggattc agtgggatac ttttgtactt caccagagcc 4321 atgttagaaa tctgtcccag catgattgga caggtgcaca gccaggaggc cattcggctc 4381 caaggaaaga atctgccgtc cctcatctgc agggttccgt tgccagtact ggccagtgca 4441 taagtgatag attcgacaaa gggcaggtac acggactccc aagggacctt gggagccctg 4501 ctaatcatga agattgtctg gcagcctgag accacggccc acaccagctg cagcagtgag 4561 acgaaggtgg agtcgattct ggacatattg tagcactcgg tgtgagttga tgcccattcc 4621 ccatagacga cctgtgatgc catggatcct agcataactt cgtataaagt atcctatacg 4681 aagttatttg ccttaaccca gaaattatca ctgttattct ttagaatggt gcaaagaata 4741 acttcgtata atgtatgcta tacgaagtta tgaattcgat atcaagctta tcgataatca 4801 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt 4861 tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc 4921 tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc 4981 cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg 5041 gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc 5101 cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg 5161 cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgccta 5221 tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc 5281 agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct 5341 tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatcgat accgagcgct 5401 gctcgagaga tctacgggtg gcatccctgt gacccctccc cagtgcctct cctggccctg 5461 gaagttgcca ctccagtgcc caccagcctt gtcctaataa aattaagttg catcattttg 5521 tctgactagg tgtccttcta taatattatg gggtggaggg gggtggtatg gagcaagggg 5581 caagttggga agacaacctg tagggcctgc ggggtctatt gggaaccaag ctggagtgca 5641 gtggcacaat cttggctcac tgcaatctcc gcctcctggg ttcaagcgat tctcctgcct 5701 cagcctcccg agttgttggg attccaggca tgcatgacca ggctcagcta atttttgttt 5761 ttttggtaga gacggggttt caccatattg gccaggctgg tctccaactc ctaatctcag 5821 gtgatctacc caccttggcc tcccaaattg ctgggattac aggcgtgaac cactgctccc 5881 ttccctgtcc ttctgatttt gtaggtaacc acgtgcggac cgagcggccg caggaacccc 5941 tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac 6001 caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca 6061 gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac 6121 accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg 6181 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt 6241 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg 6301 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga 6361 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac 6421 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc 6481 tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct attggttaaa 6541 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat 6601 tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca 6661 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag 6721 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa 6781 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat 6841 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 6901 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 6961 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 7021 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 7081 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 7141 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 7201 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg 7261 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 7321 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 7381 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 7441 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 7501 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact 7561 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 7621 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 7681 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 7741 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 7801 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 7861 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 7921 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 7981 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 8041 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 8101 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 8161 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 8221 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 8281 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 8341 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 8401 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 8461 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 8521 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 8581 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 8641 ggccttttgc tggccttttg ctcacatgt //