LOCUS       sequence-299724-        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        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      661..670
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     sig_peptide     667..726
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     CDS             730..756
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             763..1719
                     /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             1726..1767
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1771..1917
                     /codon_start=1
                     /product="transmembrane domain from platelet derived growth
                     factor receptor beta"
                     /label=PDGFR-beta TM domain
                     /translation="AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQ
                     KKPR"
     promoter        complement(1948..1966)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1949..1966)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1986..2003)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1992..2216
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2262..2690
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2349..2368)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2559..2580
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2699..2719)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2704..3033
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2884..3019
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2946..2965
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3100..3894
                     /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(3154..3173)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3764..3783
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4068..4189
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4105..4124)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4159..4178
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4238..4254)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4238..4254)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4251..4273)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4262..4278
                     /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(4286..4292,4293..4310,4311..4316))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4331..4352
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4469..4486)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4640..5228)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4720..4739)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5399..6190,6191..6259))
                     /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(6260..6364)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6439..6458)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      661 gccaccatgg agacagacac actcctgcta tgggtgctgc tgctctgggt tccaggttcc
      721 actggtgact acccatacga tgttccagat tacgctgcta gcaaagacaa tactgtgcct
      781 ctgaagctga tcgctctcct ggctaatggc gagttccata gtggcgaaca gctgggagaa
      841 accctgggca tgtccagggc cgctatcaac aagcacattc agactctgcg cgactggggc
      901 gtggacgtgt tcaccgtgcc cggaaagggc tactctctgc ccgagcctat cccgctgctg
      961 aacgctaaac agattctggg acagctggac ggcgggagcg tggcagtcct gcctgtggtc
     1021 gactccacca atcagtacct gctggatcga atcggcgagc tgaagagtgg ggatgcttgc
     1081 attgcagaat atcagcaggc agggagagga agcagaggga ggaaatggtt ctctcctttt
     1141 ggagctaacc tgtacctgag tatgttttgg cgcctgaagc ggggaccagc agcaatcggc
     1201 ctgggcccgg tcatcggaat tgtcatggca gaagcgctgc gaaagctggg agcagacaag
     1261 gtgcgagtca aatggcccaa tgacctgtat ctgcaggata gaaagctggc aggcatcctg
     1321 gtggagctgg ccggaataac aggcgatgct gcacagatcg tcattggcgc cgggattaac
     1381 gtggctatga ggcgcgtgga ggaaagcgtg gtcaatcagg gctggatcac actgcaggaa
     1441 gcagggatta acctggacag gaatactctg gccgctacgc tgatccgaga gctgcgggca
     1501 gccctggaac tgttcgagca ggaaggcctg gctccatatc tgccacggtg ggagaagctg
     1561 gataacttca tcaatagacc cgtgaagctg atcattgggg acaaagagat tttcgggatt
     1621 agccggggga ttgataaaca gggagccctg ctgctggaac aggacggagt tatcaaaccc
     1681 tggatgggcg gagaaatcag tctgcggtct gccgaaaagc tgcagggcaa gcccatcccc
     1741 aaccccctgc tgggcctgga cagcaccaat gctgtgggcc aggacacgca ggaggtcatc
     1801 gtggtgccac actccttgcc ctttaaggtg gtggtgatct cagccatcct ggccctggtg
     1861 gtgctcacca tcatctccct tatcatcctc atcatgcttt ggcagaagaa gccacgttag
     1921 ctcgagcatg catctagagg gccctattct atagtgtcac ctaaatgcta gagctcgctg
     1981 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     2041 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     2101 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     2161 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
     2221 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
     2281 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     2341 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     2401 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     2461 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     2521 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     2581 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     2641 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg
     2701 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     2761 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     2821 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     2881 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     2941 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
     3001 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt
     3061 ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     3121 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     3181 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     3241 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
     3301 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     3361 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     3421 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     3481 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     3541 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     3601 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc
     3661 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     3721 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     3781 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     3841 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     3901 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     3961 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     4021 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     4081 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     4141 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     4201 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     4261 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     4321 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     4381 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4441 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4501 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4561 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4621 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4681 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4741 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4801 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4861 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacaa accccccgtt cagcccgacc
     4921 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4981 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5041 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     5101 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5161 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5221 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5281 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5341 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5401 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5461 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5521 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5581 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5641 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5701 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5761 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5821 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     5881 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     5941 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     6001 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6061 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6121 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6181 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6241 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6301 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6361 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc
     6421 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
     6481 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
     6541 gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
     6601 tcgcgatgta cgggccagat atacgcgtt
//
