LOCUS Exported 5947 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Hepatocyte-specific expression of cytosolic TurboID, N-term V5 tag, in AAV expression plasmid. Amp selection. ACCESSION . VERSION . KEYWORDS pAAV-TBG-Cyto-TurboID SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5947) 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 5947) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5947 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind 49..70 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 85..115 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 123..139 /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 128..150 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 147..163 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 147..163 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" repeat_region 201..330 /label=AAV2 ITR promoter 670..1079 /label=TBG promoter /note="promoter from the human thyroxine-binding globulin gene (Yan et al., 2012)" intron 1139..1271 /label=chimeric intron /note="chimera between introns from human beta-globin and immunoglobulin heavy chain genes" primer_bind 1254..1273 /label=pMT2-F /note="Synthetic intron, forward primer" CDS 1291..1332 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" CDS 1339..2295 /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 2302..2328 /codon_start=1 /product="nuclear export signal from the HIV Rev protein (Fischer et al., 1995)" /label=NES /translation="LPPLERLTL" primer_bind complement(2400..2420) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(2818..2829) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(2896..2913) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2902..3109 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 3197..3326 /label=AAV2 ITR primer_bind complement(3345..3362) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(3345..3361) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3354..3376) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 3503..3958 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3590..3609) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3800..3821 /label=F1ori-F /note="F1 origin, forward primer" promoter 3984..4088 /gene="bla" /label=AmpR promoter CDS 4089..4949 /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(4307..4326) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5120..5708 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5609..5628 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5862..5879 /label=L4440 /note="L4440 vector, forward primer" ORIGIN 1 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta 61 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg 121 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccagatt 181 taattaaggc cttaattagg ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc 241 ccgggcgtcg ggcgaccttt ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg 301 gagtggccaa ctccatcact aggggttcct tgtagttaat gattaacccg ccatgctact 361 tatctacgta gccatgctct aggaagatcg gaattcgccc ttaagctagc aggttaattt 421 ttaaaaagca gtcaaaagtc caagtggccc ttggcagcat ttactctctc tgtttgctct 481 ggttaataat ctcaggagca caaacattcc agatccaggt taatttttaa aaagcagtca 541 aaagtccaag tggcccttgg cagcatttac tctctctgtt tgctctggtt aataatctca 601 ggagcacaaa cattccagat ccggcgcgcc agggctggaa gctacctttg acatcatttc 661 ctctgcgaat gcatgtataa tttctacaga acctattaga aaggatcacc cagcctctgc 721 ttttgtacaa ctttccctta aaaaactgcc aattccactg ctgtttggcc caatagtgag 781 aactttttcc tgctgcctct tggtgctttt gcctatggcc cctattctgc ctgctgaaga 841 cactcttgcc agcatggact taaacccctc cagctctgac aatcctcttt ctcttttgtt 901 ttacatgaag ggtctggcag ccaaagcaat cactcaaagt tcaaacctta tcattttttg 961 ctttgttcct cttggccttg gttttgtaca tcagctttga aaataccatc ccagggttaa 1021 tgctggggtt aatttataac taagagtgct ctagttttgc aatacaggac atgctataaa 1081 aatggaaaga tgttgctttc tgagagactg cagaagttgg tcgtgaggca ctgggcaggt 1141 aagtatcaag gttacaagac aggtttaagg agaccaatag aaactgggct tgtcgagaca 1201 gagaagactc ttgcgtttct gataggcacc tattggtctt actgacatcc actttgcctt 1261 tctctccaca ggtgtccagg cggccgcatg ggcaagccca tccccaaccc cctgctgggc 1321 ctggacagca ccgctagcaa agacaatact gtgcctctga agctgatcgc tctcctggct 1381 aatggcgagt tccatagtgg cgaacagctg ggagaaaccc tgggcatgtc cagggccgct 1441 atcaacaagc acattcagac tctgcgcgac tggggcgtgg acgtgttcac cgtgcccgga 1501 aagggctact ctctgcccga gcctatcccg ctgctgaacg ctaaacagat tctgggacag 1561 ctggacggcg ggagcgtggc agtcctgcct gtggtcgact ccaccaatca gtacctgctg 1621 gatcgaatcg gcgagctgaa gagtggggat gcttgcattg cagaatatca gcaggcaggg 1681 agaggaagca gagggaggaa atggttctct ccttttggag ctaacctgta cctgagtatg 1741 ttttggcgcc tgaagcgggg accagcagca atcggcctgg gcccggtcat cggaattgtc 1801 atggcagaag cgctgcgaaa gctgggagca gacaaggtgc gagtcaaatg gcccaatgac 1861 ctgtatctgc aggatagaaa gctggcaggc atcctggtgg agctggccgg aataacaggc 1921 gatgctgcac agatcgtcat tggcgccggg attaacgtgg ctatgaggcg cgtggaggaa 1981 agcgtggtca atcagggctg gatcacactg caggaagcag ggattaacct ggacaggaat 2041 actctggccg ctacgctgat ccgagagctg cgggcagccc tggaactgtt cgagcaggaa 2101 ggcctggctc catatctgcc acggtgggag aagctggata acttcatcaa tagacccgtg 2161 aagctgatca ttggggacaa agagattttc gggattagcc gggggattga taaacaggga 2221 gccctgctgc tggaacagga cggagttatc aaaccctgga tgggcggaga aatcagtctg 2281 cggtctgccg aaaagctgca gctgcctccc ctggagcgcc tgaccctgga ctaaaagctt 2341 ggatccaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat 2401 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct 2461 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag 2521 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc 2581 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc 2641 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct 2701 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg 2761 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg 2821 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg 2881 cgtcttcgag atctgcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct 2941 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg 3001 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc 3061 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggac tcgagttaag 3121 ggcgaattcc cgataaggat cttcctagag catggctacg tagataagta gcatggcggg 3181 ttaatcatta actacaagga acccctagtg atggagttgg ccactccctc tctgcgcgct 3241 cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg 3301 gcctcagtga gcgagcgagc gcgcagcctt aattaaccta attcactggc cgtcgtttta 3361 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc 3421 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg 3481 cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg 3541 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct 3601 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg 3661 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag 3721 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg 3781 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc 3841 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat 3901 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag 3961 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 4021 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 4081 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 4141 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 4201 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 4261 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 4321 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 4381 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 4441 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 4501 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 4561 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 4621 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 4681 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 4741 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 4801 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 4861 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 4921 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 4981 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 5041 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 5101 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 5161 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 5221 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc 5281 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 5341 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 5401 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 5461 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 5521 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 5581 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 5641 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 5701 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 5761 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 5821 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 5881 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 5941 gcagctg //