LOCUS Exported 7086 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Cre recombinase-dependent expression of ER TurboID, N-term HA tag, C-term V5 tag, in AAV expression plasmid. Amp selection. ACCESSION . VERSION . KEYWORDS pAAV-FLEx-ER-TurboID SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7086) AUTHORS Wei W, Riley NM, Yang AC, Kim JT, Terrell SM, Li VL, Garcia-Contreras M, Bertozzi CR, Long JZ. TITLE Cell type-selective secretome profiling in vivo JOURNAL Nature Chemical Biology. 2020 REFERENCE 2 (bases 1 to 7086) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7086 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..130 /label=AAV2 ITR enhancer 204..583 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 585..862 /label=chicken beta-actin promoter intron 863..1879 /label=chimeric intron /note="chimera between introns from chicken beta-actin and rabbit beta-globin" primer_bind 1803..1825 /label=pCAGGS-5 /note="Chimeric intron in CAG promoter, forward primer" primer_bind 1887..1906 /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" protein_bind 1934..1967 /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 2018..2051 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS complement(2072..2113) /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" CDS complement(2120..3076) /codon_start=1 /product="mutant of the E. coli biotin ligase BioID engineered for improved catalytic efficiency (Branon et al., 2018)" /label=TurboID /translation="KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDW GVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGD ACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLG ADKVRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWI TLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDK EIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK" CDS complement(3083..3109) /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" sig_peptide complement(3110..3172) /product="leader sequence from mouse immunoglobulin kappa light chain" /label=Ig-kappa leader regulatory 3169..3178 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" protein_bind complement(3179..3212) /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(3263..3296) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 3309..3897 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(3362..3382) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3780..3791) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(3923..3942) /label=Bglob-pA-R /note="Rabbit beta-globin polyA region, reverse primer" polyA_signal 3988..4043 /label=-beta-globin poly(A) signal /note="rabbit beta-globin polyadenylation signal (Gil and Proudfoot, 1987)" repeat_region 4136..4265 /label=AAV2 ITR primer_bind complement(4284..4301) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(4284..4300) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4293..4315) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 4442..4897 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4529..4548) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4739..4760 /label=F1ori-F /note="F1 origin, forward primer" promoter 4923..5027 /gene="bla" /label=AmpR promoter CDS 5028..5888 /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(5246..5265) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6059..6647 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6548..6567 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6801..6818 /label=L4440 /note="L4440 vector, forward primer" protein_bind 6935..6956 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 6971..7001 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 7009..7025 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." primer_bind 7014..7036 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 7033..7049 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 7033..7049 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" ORIGIN 1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 121 aggggttcct tgtagttaat gattaacccg ccatgctact tatctaccag ggtaatgggg 181 atcctctaga actatagcta gtcgacattg attattgact agttattaat agtaatcaat 241 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa 301 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt 361 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta 421 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt 481 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc 541 tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac 601 gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat 661 tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccrggggg 721 gggggggsgg ggsgrggggs ggggsggggc gaggcggaga ggtgcggcgg cagccaatca 781 gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa 841 aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc 901 cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag 961 cgggcgggac ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggcttgt 1021 ttcttttctg tggctgcgtg aaagccttga ggggctccgg gagggccctt tgtgcggggg 1081 gagcggctcg gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc 1141 gctgcccggc ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt 1201 gcgcgagggg agcgcggccg ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa 1261 caaaggctgc gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt 1321 cgggctgcaa ccccccctgc acccccctcc ccgagttgct gagcacggcc cggcttcggg 1381 tgcggggctc cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca 1441 ggtgggggtg ccgggcgggg cggggccgcc tcgggccggg grgggctcgg gggaggggcg 1501 cggcggcccc cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc attgcctttt 1561 atggtaatcg tgcgagaggg cgcagggact tcctttgtcc caaatctgtg cggagccgaa 1621 atctgggagg cgccgccgca ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg 1681 caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc cccttctccc 1741 tctccagcct cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc 1801 ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc 1861 cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt 1921 tggcaaagaa ttcataactt cgtataggat actttatacg aagttatgca gaatggtagc 1981 tggattgtag ctgctattag caatatgaaa cctcttaata acttcgtata gcatacatta 2041 tacgaagtta ttgcagttag agttcatctt tggtgctgtc caggcccagc agggggttgg 2101 ggatgggctt gccctgcagc ttttcggcag accgcagact gatttctccg cccatccagg 2161 gtttgataac tccgtcctgt tccagcagca gggctccctg tttatcaatc ccccggctaa 2221 tcccgaaaat ctctttgtcc ccaatgatca gcttcacggg tctattgatg aagttatcca 2281 gcttctccca ccgtggcaga tatggagcca ggccttcctg ctcgaacagt tccagggctg 2341 cccgcagctc tcggatcagc gtagcggcca gagtattcct gtccaggtta atccctgctt 2401 cctgcagtgt gatccagccc tgattgacca cgctttcctc cacgcgcctc atagccacgt 2461 taatcccggc gccaatgacg atctgtgcag catcgcctgt tattccggcc agctccacca 2521 ggatgcctgc cagctttcta tcctgcagat acaggtcatt gggccatttg actcgcacct 2581 tgtctgctcc cagctttcgc agcgcttctg ccatgacaat tccgatgacc gggcccaggc 2641 cgattgctgc tggtccccgc ttcaggcgcc aaaacatact caggtacagg ttagctccaa 2701 aaggagagaa ccatttcctc cctctgcttc ctctccctgc ctgctgatat tctgcaatgc 2761 aagcatcccc actcttcagc tcgccgattc gatccagcag gtactgattg gtggagtcga 2821 ccacaggcag gactgccacg ctcccgccgt ccagctgtcc cagaatctgt ttagcgttca 2881 gcagcgggat aggctcgggc agagagtagc cctttccggg cacggtgaac acgtccacgc 2941 cccagtcgcg cagagtctga atgtgcttgt tgatagcggc cctggacatg cccagggttt 3001 ctcccagctg ttcgccacta tggaactcgc cattagccag gagagcgatc agcttcagag 3061 gcacagtatt gtctttgcta gcagcgtaat ctggaacatc gtatgggtag tcaccagtgg 3121 aacctggaac ccagagcagc agcacccata gcaggagtgt gtctgtctcc atggtggcat 3181 aacttcgtat aaagtatcct atacgaagtt atttgcctta acccagaaat tatcactgtt 3241 attctttaga atggtgcaaa gaataacttc gtataatgta tgctatacga agttataagc 3301 ttatcgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact 3361 atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg 3421 cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg 3481 aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa 3541 cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc 3601 ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg 3661 ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt 3721 ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt 3781 cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc 3841 cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc 3901 gataccgtcg atgaggatcc gatctttttc cctctgccaa aaattatggg gacatcatga 3961 agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt 4021 gttggaattt tttgtgtctc tcactcggaa gcaattcgtt gatctgaatt tcgaccaccc 4081 ataataccca ttaccctggt agataagtag catggcgggt taatcattaa ctacaaggaa 4141 cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg 4201 cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg 4261 cgcagcctta attaacctaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac 4321 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat 4381 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg 4441 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 4501 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 4561 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 4621 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg 4681 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 4741 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta 4801 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 4861 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg 4921 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 4981 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac 5041 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc 5101 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca 5161 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc 5221 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg 5281 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac 5341 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca 5401 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg 5461 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac 5521 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg 5581 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat 5641 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg 5701 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg 5761 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc 5821 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc 5881 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt 5941 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt 6001 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt 6061 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag 6121 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca 6181 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca 6241 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg 6301 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg 6361 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct 6421 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga 6481 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc 6541 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg 6601 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg 6661 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt 6721 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc 6781 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac 6841 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc 6901 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg 6961 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat 7021 aacaatttca cacaggaaac agctatgacc atgattacgc cagatttaat taaggcctta 7081 attagg //