LOCUS       sequence-292035-        6629 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of secretory pathway membrane-localized 
            TurboID, N-term HA tag, C-term V5 tag, in mammalian expression 
            plasmid. Amp/Neo selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6629)
  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 6629)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 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: pcDNA3-mem-TurboID
FEATURES             Location/Qualifiers
     source          1..6629
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        145..524
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        525..728
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     679..699
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     773..792
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        773..791
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      805..814
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     sig_peptide     811..870
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     CDS             874..900
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             907..1863
                     /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             1870..1911
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1915..2061
                     /codon_start=1
                     /product="transmembrane domain from platelet derived growth
                     factor receptor beta"
                     /label=PDGFR-beta TM domain
                     /translation="AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQ
                     KKPR"
     promoter        complement(2092..2110)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2093..2110)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2130..2147)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2136..2360
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2406..2834
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2493..2512)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2703..2724
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2843..2863)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2848..3177
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3028..3163
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3090..3109
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3244..4038
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3298..3317)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3908..3927
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4212..4333
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4249..4268)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4303..4322
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4382..4398)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4382..4398)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4395..4417)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4406..4422
                     /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)."
     promoter        complement(join(4430..4436,4437..4454,4455..4460))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4475..4496
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4613..4630)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4784..5372)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4864..4883)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5543..6334,6335..6403))
                     /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"
     promoter        complement(6404..6508)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6583..6602)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag
       61 gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg
      121 atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa
      181 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
      241 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
      301 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
      361 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
      421 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
      481 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
      541 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
      601 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
      661 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
      721 gcagagctct ctggctaact agagaaccca ctgcttactg gcttatcgaa attaatacga
      781 ctcactatag ggagacccaa gcttgccacc atggagacag acacactcct gctatgggtg
      841 ctgctgctct gggttccagg ttccactggt gactacccat acgatgttcc agattacgct
      901 gctagcaaag acaatactgt gcctctgaag ctgatcgctc tcctggctaa tggcgagttc
      961 catagtggcg aacagctggg agaaaccctg ggcatgtcca gggccgctat caacaagcac
     1021 attcagactc tgcgcgactg gggcgtggac gtgttcaccg tgcccggaaa gggctactct
     1081 ctgcccgagc ctatcccgct gctgaacgct aaacagattc tgggacagct ggacggcggg
     1141 agcgtggcag tcctgcctgt ggtcgactcc accaatcagt acctgctgga tcgaatcggc
     1201 gagctgaaga gtggggatgc ttgcattgca gaatatcagc aggcagggag aggaagcaga
     1261 gggaggaaat ggttctctcc ttttggagct aacctgtacc tgagtatgtt ttggcgcctg
     1321 aagcggggac cagcagcaat cggcctgggc ccggtcatcg gaattgtcat ggcagaagcg
     1381 ctgcgaaagc tgggagcaga caaggtgcga gtcaaatggc ccaatgacct gtatctgcag
     1441 gatagaaagc tggcaggcat cctggtggag ctggccggaa taacaggcga tgctgcacag
     1501 atcgtcattg gcgccgggat taacgtggct atgaggcgcg tggaggaaag cgtggtcaat
     1561 cagggctgga tcacactgca ggaagcaggg attaacctgg acaggaatac tctggccgct
     1621 acgctgatcc gagagctgcg ggcagccctg gaactgttcg agcaggaagg cctggctcca
     1681 tatctgccac ggtgggagaa gctggataac ttcatcaata gacccgtgaa gctgatcatt
     1741 ggggacaaag agattttcgg gattagccgg gggattgata aacagggagc cctgctgctg
     1801 gaacaggacg gagttatcaa accctggatg ggcggagaaa tcagtctgcg gtctgccgaa
     1861 aagctgcagg gcaagcccat ccccaacccc ctgctgggcc tggacagcac caatgctgtg
     1921 ggccaggaca cgcaggaggt catcgtggtg ccacactcct tgccctttaa ggtggtggtg
     1981 atctcagcca tcctggccct ggtggtgctc accatcatct cccttatcat cctcatcatg
     2041 ctttggcaga agaagccacg ttagctcgag catgcatcta gagggcccta ttctatagtg
     2101 tcacctaaat gctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     2161 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     2221 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     2281 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     2341 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta
     2401 tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     2461 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     2521 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     2581 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     2641 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     2701 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     2761 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     2821 atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     2881 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     2941 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     3001 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     3061 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc
     3121 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     3181 ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt
     3241 cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
     3301 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg
     3361 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
     3421 ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct
     3481 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg
     3541 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
     3601 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
     3661 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
     3721 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc
     3781 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa
     3841 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
     3901 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
     3961 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
     4021 cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca
     4081 acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     4141 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     4201 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     4261 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     4321 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
     4381 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     4441 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     4501 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     4561 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     4621 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     4681 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     4741 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     4801 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     4861 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     4921 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     4981 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     5041 acaaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     5101 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     5161 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     5221 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     5281 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     5341 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     5401 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     5461 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     5521 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     5581 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     5641 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     5701 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     5761 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     5821 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     5881 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     5941 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     6001 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     6061 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     6121 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     6181 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     6241 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     6301 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     6361 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     6421 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     6481 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg
     6541 acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc
     6601 cgcatagtta agccagtatc tgctccctg
//
