LOCUS       sequence-292040-        6160 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Hepatocyte-specific expression of secretory pathway 
            membrane-localized TurboID, N-term HA tag, C-term V5 tag, in AAV 
            expression plasmid. Amp selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6160)
  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   Nat Chem Biol. 2021 Mar;17(3):326-334. doi: 
            10.1038/s41589-020-00698-y. Epub 2020 Nov 16.
  PUBMED    33199915
REFERENCE   2  (bases 1 to 6160)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41589-020-00698-y"; journalName: "Nat Chem Biol"; date: 
            "2021-03"; volume: "17"; issue: "3"; pages: "326-334"
COMMENT     Sequence Label: pAAV-TBG-mem-TurboID
FEATURES             Location/Qualifiers
     source          1..6160
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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        join(85..90,91..108,109..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"
     regulatory      1288..1297
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     sig_peptide     1294..1356
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTGD"
     CDS             1357..1383
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1390..2346
                     /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             2353..2394
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2398..2544
                     /codon_start=1
                     /product="transmembrane domain from platelet derived growth
                     factor receptor beta"
                     /label=PDGFR-beta TM domain
                     /translation="AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQ
                     KKPR"
     primer_bind     complement(2613..2633)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3031..3042)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3109..3126)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3115..3322
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3410..3539
                     /label=AAV2 ITR
     primer_bind     complement(3558..3575)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3558..3574)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3567..3589)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3716..4171
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3803..3822)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4013..4034
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4197..4301
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4302..4370,4371..5162)
                     /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"
     rep_origin      5333..5921
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5822..5841
                     /label=pBR322ori-F
                     /note="pBR322 origin, 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 cggccgcgcc accatggaga cagacacact cctgctatgg
     1321 gtgctgctgc tctgggttcc aggttccact ggtgactacc catacgatgt tccagattac
     1381 gctgctagca aagacaatac tgtgcctctg aagctgatcg ctctcctggc taatggcgag
     1441 ttccatagtg gcgaacagct gggagaaacc ctgggcatgt ccagggccgc tatcaacaag
     1501 cacattcaga ctctgcgcga ctggggcgtg gacgtgttca ccgtgcccgg aaagggctac
     1561 tctctgcccg agcctatccc gctgctgaac gctaaacaga ttctgggaca gctggacggc
     1621 gggagcgtgg cagtcctgcc tgtggtcgac tccaccaatc agtacctgct ggatcgaatc
     1681 ggcgagctga agagtgggga tgcttgcatt gcagaatatc agcaggcagg gagaggaagc
     1741 agagggagga aatggttctc tccttttgga gctaacctgt acctgagtat gttttggcgc
     1801 ctgaagcggg gaccagcagc aatcggcctg ggcccggtca tcggaattgt catggcagaa
     1861 gcgctgcgaa agctgggagc agacaaggtg cgagtcaaat ggcccaatga cctgtatctg
     1921 caggatagaa agctggcagg catcctggtg gagctggccg gaataacagg cgatgctgca
     1981 cagatcgtca ttggcgccgg gattaacgtg gctatgaggc gcgtggagga aagcgtggtc
     2041 aatcagggct ggatcacact gcaggaagca gggattaacc tggacaggaa tactctggcc
     2101 gctacgctga tccgagagct gcgggcagcc ctggaactgt tcgagcagga aggcctggct
     2161 ccatatctgc cacggtggga gaagctggat aacttcatca atagacccgt gaagctgatc
     2221 attggggaca aagagatttt cgggattagc cgggggattg ataaacaggg agccctgctg
     2281 ctggaacagg acggagttat caaaccctgg atgggcggag aaatcagtct gcggtctgcc
     2341 gaaaagctgc agggcaagcc catccccaac cccctgctgg gcctggacag caccaatgct
     2401 gtgggccagg acacgcagga ggtcatcgtg gtgccacact ccttgccctt taaggtggtg
     2461 gtgatctcag ccatcctggc cctggtggtg ctcaccatca tctcccttat catcctcatc
     2521 atgctttggc agaagaagcc acgttaaaag cttggatcca atcaacctct ggattacaaa
     2581 atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac
     2641 gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc
     2701 ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt
     2761 ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc
     2821 tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc
     2881 gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg
     2941 gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt
     3001 ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc
     3061 cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gagatctgcc tcgactgtgc
     3121 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     3181 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     3241 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     3301 acaatagcag gcatgctggg gactcgagtt aagggcgaat tcccgataag gatcttccta
     3361 gagcatggct acgtagataa gtagcatggc gggttaatca ttaactacaa ggaaccccta
     3421 gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca
     3481 aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc
     3541 cttaattaac ctaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     3601 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     3661 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg
     3721 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     3781 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     3841 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     3901 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     3961 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     4021 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     4081 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     4141 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
     4201 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     4261 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     4321 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     4381 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     4441 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     4501 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     4561 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     4621 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     4681 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     4741 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     4801 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     4861 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     4921 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     4981 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     5041 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     5101 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     5161 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     5221 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     5281 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     5341 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     5401 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     5461 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     5521 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     5581 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     5641 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     5701 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     5761 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     5821 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     5881 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     5941 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     6001 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     6061 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
     6121 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
//
