LOCUS Exported 6217 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION An AAV vector that expresses myristoylated TagRFP-T and post-eGRASP linked by self-cleaving P2A peptide under the tetO promoter.. ACCESSION . VERSION . KEYWORDS pAAV-TIWB-myrTagRFP-T-P2A-post-eGRASP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6217) AUTHORS Choi JH, Sim SE, Kim JI, Choi DI, Oh J, Ye S, Lee J, Kim T, Ko HG, Lim CS, Kaang BK TITLE Interregional synaptic maps among engram cells underlie memory formation. JOURNAL Science. 2018 Apr 27;360(6387):430-435. doi: 10.1126/science.aas9204. PUBMED 29700265 REFERENCE 2 (bases 1 to 6217) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6217 /organism="synthetic DNA construct" /mol_type="other DNA" intron 1..133 /label=chimeric intron /note="chimera between introns from human beta-globin and immunoglobulin heavy chain genes" primer_bind 116..135 /label=pMT2-F /note="Synthetic intron, forward primer" promoter 178..196 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 200..209 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 257..982 /codon_start=1 /product="photostable variant of TagRFP (Shaner et al., 2008)" /label=TagRFP-T /translation="SKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRI KVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVL TATQDTSFQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTDMA LKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVAR YCDLPSKLGHKLNGMDELYK" CDS 992..1048 /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 1337..1384 /codon_start=1 /product="11th beta-strand of superfolder GFP" /label=GFP11 /note="associates with GFP(1-10), consisting of the first 10 beta-strands, to reconstitute fluorescent GFP (Cabantous et al., 2005; Kamiyama et al., 2016)" /translation="RDHMVLHEYVNAAGIT" CDS complement(1679..1690) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" misc_feature 2076..2664 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(2129..2149) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(2547..2558) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 2677..2901 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 2909..3049 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 2909..3038 /label=AAV2 ITR rep_origin 3124..3579 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3211..3230) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3421..3442 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3596..3615) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3715..3737 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(3775..3793) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 3861..3965 /gene="bla" /label=AmpR promoter CDS 3966..4826 /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(4184..4203) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 4997..5585 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5486..5505 /label=pBR322ori-F /note="pBR322 origin, forward primer" repeat_region 5647..5776 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" protein_bind 5800..5818 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 5836..5854 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 5872..5890 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 5908..5926 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 5944..5962 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 5980..5998 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 6016..6034 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 6079..6103 /label=LNCX /note="Human CMV promoter, forward primer" ORIGIN 1 gtaagtatca aggttacaag acaggtttaa ggagaccaat agaaactggg cttgtcgaga 61 cagagaagac tcttgcgttt ctgataggca cctattggtc ttactgacat ccactttgcc 121 tttctctcca caggtgtcca ctcccagttc aattacagct cttaaggcta gagtacttaa 181 tacgactcac tataggctag ccaccatggg ctgtgtgcag tgtaaggaca aggaggctac 241 caagctgaca ggaggctcta agggcgaaga gctgattaag gagaacatgc acatgaagct 301 gtacatggag ggcaccgtga acaaccacca cttcaagtgc acatccgagg gcgaaggcaa 361 gccctacgag ggcacccaga ccatgagaat caaggtggtc gagggcggcc ctctcccctt 421 cgccttcgac atcctggcta ccagcttcat gtacggcagc agaaccttca tcaaccacac 481 ccagggcatc cccgacttct ttaagcagtc cttccctgag ggcttcacat gggagagagt 541 caccacatac gaagacgggg gcgtgctgac cgctacccag gacaccagct tccaggacgg 601 ctgcctcatc tacaacgtca agatcagagg ggtgaacttc ccatccaacg gccctgtgat 661 gcagaagaaa acactcggct gggaggccaa caccgagatg ctgtaccccg ctgacggcgg 721 cctggaaggc agaaccgaca tggccctgaa gctcgtgggc gggggccacc tgatctgcaa 781 cttcaagacc acatacagat ccaagaaacc cgctaagaac ctcaagatgc ccggcgtcta 841 ctatgtggac cacagactgg aaagaatcaa ggaggccgac aaagagacct acgtcgagca 901 gcacgaggtg gctgtggcca gatactgcga cctccctagc aaactggggc acaaacttaa 961 tggcatggac gagctgtaca agggaagcgg agctactaac ttcagcctgc tgaagcaggc 1021 tggagacgtg gaggagaacc ctggacctga gaccgacacc ctcctgctat gggtgctgct 1081 gctctgggtg cccggcagca ccggcgacgc accggtcgga ggcaacgacc ctaacctgtt 1141 cgtcgccctg tacgacttcg tcgcctctgg cgacaacacc ctgagcatca ccaagggcga 1201 gaagctgaga gtcctgggct acaaccacaa cggcgagtgg tgcgaggccc agaccaagaa 1261 cggccagggc tgggtcccta gcaactacat cacacctgtc aacagcaccg gtggaggcag 1321 cggaggaggc tccggaagag accacatggt gctgcacgag tacgtgaacg ctgctggaat 1381 cacaggagga ggctccggag gcggaagcgg taccctcgag ctggtgcccc acttgcataa 1441 cctcaatgac atcagccaat acacaagtac aaccaccaag gtcccaagca ctgacatcac 1501 ccttcgacca actcggaaaa actcaacccc tgtgaccagc gcatttccaa ccgctaagca 1561 agacgacccc aaacaacagc ctagtccatt tagcgtagac caaagggact acagcacaga 1621 gctgtccgtg accatagctg taggagcttc cctgcttttc ctgaacattc tggctttcgc 1681 ggccctctat tacaagaagg acaaaaggcg ccacgacgta cataggagat gtagtccgca 1741 acgaactaca accaatgatc ttacgcacgc acctgaggag gagatcatga gtcttcagat 1801 gaaacacaca gacctggacc acgagtgtga atccatccat ccccatgagg tggtgctgag 1861 gacagcatgc cctccggact ataccctggc aatgcgccga agcccagacg acattcccct 1921 gatgacaccc aataccataa ccatgattcc caacactatt ccaggaatcc agcctctcca 1981 cactttcaac acattcactg gtggacagaa taatactctc cctcatccgc atccacaccc 2041 ccactcccac agctaagaat tcctgcagcc ccgataatca acctctggat tacaaaattt 2101 gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg 2161 ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt 2221 ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg 2281 tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc 2341 agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg 2401 cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt 2461 tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc 2521 gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg 2581 gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga 2641 tctccctttg ggccgcctcc ccgcatcggg gggatcctgt gccttctagt tgccagccat 2701 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc 2761 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg 2821 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg 2881 gggatgcggt gggctctatg gcggccgcag gaacccctag tgatggagtt ggccactccc 2941 tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc 3001 tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg 3061 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat 3121 agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 3181 ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg 3241 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat 3301 ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg 3361 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata 3421 gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt 3481 tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat 3541 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa 3601 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc 3661 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga 3721 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg 3781 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg 3841 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa 3901 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 3961 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc 4021 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg 4081 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc 4141 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat 4201 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 4261 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag 4321 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 4381 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc 4441 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca 4501 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 4561 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc 4621 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 4681 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta 4741 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag 4801 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga 4861 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 4921 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 4981 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 5041 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 5101 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt 5161 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 5221 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 5281 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 5341 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 5401 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 5461 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 5521 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 5581 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 5641 acatgtcctg caggcagctg cgcgctcgct cgctcactga ggccgcccgg gcgtcgggcg 5701 acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc 5761 atcactaggg gttcctgcgg ccgcacgcgt tggctttact ccctatcagt gatagagaac 5821 gtatgaagag tttactccct atcagtgata gagaacgtat gcagacttta ctccctatca 5881 gtgatagaga acgtataagg agtttactcc ctatcagtga tagagaacgt atgaccagtt 5941 tactccctat cagtgataga gaacgtatct acagtttact ccctatcagt gatagagaac 6001 gtatatccag tttactccct atcagtgata gagaacgtat aagctttagg cgtgtacggt 6061 gggcgcctat aaaagcagag ctcgtttagt gaaccgtcag atcgcctgga gcaattccac 6121 aacacttttg tcttatacca actttccgta ccacttccta ccctcgtaaa gctagtcgaa 6181 cttaagctgc agaagttggt cgtgaggcac tgggcag //