LOCUS Exported 7253 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Forebrain principal neuron expression of oChIEF kinetic variant E163A/T199C with physically uncoupled mKate2 red fluorophore. ACCESSION . VERSION . KEYWORDS pAAV-CaMKIIa-oChIEF(E163A/T199C)-P2A-mKate2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7253) TITLE ChIEF and oChIEF AAV vectors JOURNAL Unpublished REFERENCE 2 (bases 1 to 7253) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7253 /organism="synthetic DNA construct" /mol_type="other DNA" 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 164..1452 /label=CaMKII promoter /note="promoter for the mouse calcium/calmodulin-dependent kinase II alpha subunit" regulatory 1483..1492 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2581..2637 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 2638..3333 /codon_start=1 /product="monomeric far-red fluorescent protein (Shcherbo et al., 2009)" /label=mKate2 /note="mammalian codon-optimized" /translation="VSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKA VEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMALK LVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYC DLPSKLGHR" misc_feature 3380..3968 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(3433..3453) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3851..3862) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 4000..4476 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(4199..4218) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 4516..4656 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4516..4645 /label=AAV2 ITR rep_origin 4731..5186 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4818..4837) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5028..5049 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(5203..5222) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5322..5344 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5382..5400) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5468..5572 /gene="bla" /label=AmpR promoter CDS 5573..6433 /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(5791..5810) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6604..7192 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7093..7112 /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 cgcgtttaat taacattatg gccttaggtc 181 acttcatctc catggggttc ttcttctgat tttctagaaa atgagatggg ggtgcagaga 241 gcttcctcag tgacctgccc agggtcacat cagaaatgtc agagctagaa cttgaactca 301 gattactaat cttaaattcc atgccttggg ggcatgcaag tacgatatac agaaggagtg 361 aactcattag ggcagatgac caatgagttt aggaaagaag agtccagggc agggtacatc 421 tacaccaccc gcccagccct gggtgagtcc agccacgttc acctcattat agttgcctct 481 ctccagtcct accttgacgg gaagcacaag cagaaactgg gacaggagcc ccaggagacc 541 aaatcttcat ggtccctctg ggaggatggg tggggagagc tgtggcagag gcctcaggag 601 gggccctgct gctcagtggt gacagatagg ggtgagaaag cagacagagt cattccgtca 661 gcattctggg tctgtttggt acttcttctc acgctaaggt ggcggtgtga tatgcacaat 721 ggctaaaaag cagggagagc tggaaagaaa caaggacaga gacagaggcc aagtcaacca 781 gaccaattcc cagaggaagc aaagaaacca ttacagagac tacaaggggg aagggaagga 841 gagatgaatt agcttcccct gtaaacctta gaacccagct gttgccaggg caacggggca 901 atacctgtct cttcagagga gatgaagttg ccagggtaac tacatcctgt ctttctcaag 961 gaccatccca gaatgtggca cccactagcc gttaccatag caactgcctc tttgccccac 1021 ttaatcccat cccgtctgtt aaaagggccc tatagttgga ggtgggggag gtaggaagag 1081 cgatgatcac ttgtggacta agtttgttcg catccccttc tccaaccccc tcagtacatc 1141 accctggggg aacagggtcc acttgctcct gggcccacac agtcctgcag tattgtgtat 1201 ataaggccag ggcaaagagg agcaggtttt aaagtgaaag gcaggcaggt gttggggagg 1261 cagttaccgg ggcaacggga acagggcgtt tcggaggtgg ttgccatggg gacctggatg 1321 ctgacgaagg ctcgcgaggc tgtgagcagc cacagtgccc tgctcagaag ccccaagctc 1381 gtcagtcaag ccggttctcc gtttgcactc aggagcacgg gcaggcgagt ggcccctagt 1441 tctgggggca gcgggggatc cttcgaaacc ggtgctagcg gcgccaccat ggtgagcaga 1501 agaccctggc tgctggccct ggccctggcc gtggccctgg ccgccggcag cgccggcgcc 1561 agcaccggca gcgacgccac cgtgcccgtg gccacccagg acggccccga ctacgtgttc 1621 cacagagccc acgagagaat gctgttccag accagctaca ccctggagaa caacggcagc 1681 gtgatctgca tccccaacaa cggccagtgc ttctgcctgg cctggctgaa gagtaacggc 1741 accaacgccg agaagctggc cgccaacatc ctgcagtgga tcaccttcgc cctgagcgcc 1801 ctgtgcctga tgttctacgg ctaccagacc tggaagagta cctgcggctg ggaggagatc 1861 tacgtggcca ccatcgagat gatcaagttc atcatagagt acttccacga gttcgacgag 1921 cccgccgtga tctacagcag caacggcaac aagaccgtgt ggctgagata cgccgcctgg 1981 ctgctgacct gccccgtggt cctgatccac ctgagcaacc tgaccggcct ggccaacgac 2041 tacaacaaga gaaccatggg cctgctggtg agcgacatcg gctgtatcgt gtggggcacc 2101 accgccgccc tgagcaaggg ctacgtgaga gtgatcttct tcctgatggg cctgtgctac 2161 ggcatctaca ccttcttcaa cgccgccaag gtgtacatcg aggcctacca caccgtgccc 2221 aagggcagat gcagacaggt ggtgaccggc atggcctggc tgttcttcgt gagctggggc 2281 atgttcccca tcctgttcat cctgggcccc gagggcttcg gcgtgctgag cgtgtacggc 2341 agcaccgtgg gccacaccat catcgacctg atgagcaaga actgctgggg cctgctgggc 2401 cactacctga gagtgctgat ccacgagcac atcctgatcc acggcgacat cagaaagacc 2461 accaagctga acatcggcgg caccgagatc gaggtggaga ccctggtgga ggacgaggcc 2521 gaggccggcg ccgtgaacaa gggcaccggc aagtacgaga gcagcgttaa cggaagcgga 2581 gccactaact tctccctgtt gaaacaagca ggggatgtcg aagagaatcc cgggccagtg 2641 agcgagctga ttaaggagaa catgcacatg aagctgtaca tggagggcac cgtgaacaac 2701 caccacttca agtgcacatc cgagggcgaa ggcaagccct acgagggcac ccagaccatg 2761 agaatcaagg cggtcgaggg cggccctctc cccttcgcct tcgacatcct ggctaccagc 2821 ttcatgtacg gcagcaaaac cttcatcaac cacacccagg gcatccccga cttctttaag 2881 cagtccttcc ccgagggctt cacatgggag agagtcacca catacgaaga cgggggcgtg 2941 ctgaccgcta cccaggacac cagcctccag gacggctgcc tcatctacaa cgtcaagatc 3001 agaggggtga acttcccatc caacggccct gtgatgcaga agaaaacact cggctgggag 3061 gcctccaccg agaccctgta ccccgctgac ggcggcctgg aaggcagagc cgacatggcc 3121 ctgaagctcg tgggcggggg ccacctgatc tgcaacttga agaccacata cagatccaag 3181 aaacccgcta agaacctcaa gatgcccggc gtctactatg tggacagaag actggaaaga 3241 atcaaggagg ccgacaaaga gacctacgtc gagcagcacg aggtggctgt ggccagatac 3301 tgcgacctcc ctagcaaact ggggcacaga tgagtcgacg gcgcgcccct gcagggaatt 3361 cgatatcaag cttatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg 3421 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta 3481 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct 3541 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt 3601 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac 3661 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg 3721 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc 3781 gtcctttcct tggctgctcg cctatgttgc cacctggatt ctgcgcggga cgtccttctg 3841 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct 3901 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc 3961 ctccccgcat cgataccgag cgctgctcga gagatctacg ggtggcatcc ctgtgacccc 4021 tccccagtgc ctctcctggc cctggaagtt gccactccag tgcccaccag ccttgtccta 4081 ataaaattaa gttgcatcat tttgtctgac taggtgtcct tctataatat tatggggtgg 4141 aggggggtgg tatggagcaa ggggcaagtt gggaagacaa cctgtagggc ctgcggggtc 4201 tattgggaac caagctggag tgcagtggca caatcttggc tcactgcaat ctccgcctcc 4261 tgggttcaag cgattctcct gcctcagcct cccgagttgt tgggattcca ggcatgcatg 4321 accaggctca gctaattttt gtttttttgg tagagacggg gtttcaccat attggccagg 4381 ctggtctcca actcctaatc tcaggtgatc tacccacctt ggcctcccaa attgctggga 4441 ttacaggcgt gaaccactgc tcccttccct gtccttctga ttttgtaggt aaccacgtgc 4501 ggaccgagcg gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc 4561 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg 4621 cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc 4681 ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg 4741 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc 4801 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg 4861 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg 4921 gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg 4981 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt 5041 ccaaactgga acaacactca accctatctc gggctattct tttgatttat aagggatttt 5101 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt 5161 taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc 5221 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg 5281 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca 5341 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt 5401 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg 5461 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct 5521 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat 5581 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc 5641 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg 5701 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg 5761 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga 5821 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta 5881 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc 5941 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc 6001 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg 6061 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc 6121 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca 6181 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct 6241 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat 6301 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg 6361 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat 6421 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact 6481 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 6541 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 6601 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 6661 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 6721 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca 6781 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 6841 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 6901 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 6961 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 7021 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 7081 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 7141 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 7201 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgt //