LOCUS       sequence-324138-        6393 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses ER targeted TurboID for secretome biotinylation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6393)
  AUTHORS   Kim KE, Park I, Kim J, Kang MG, Choi WG, Shin H, Kim JS, Rhee HW, 
            Suh JM
  TITLE     Dynamic tracking and identification of tissue-specific secretory 
            proteins in the circulation of live mice.
  JOURNAL   Nat Commun. 2021 Sep 1;12(1):5204. doi: 10.1038/s41467-021-25546-y.
  PUBMED    34471136
REFERENCE   2  (bases 1 to 6393)
  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"; journalName: "Nat 
            Commun."; date: "2021-09-1"; volume: "12(1):5204. doi"; pages: " 
            10.1038/s41467-021-25546-y"
COMMENT     Sequence Label: Sec61b-V5-TurboID
FEATURES             Location/Qualifiers
     source          1..6393
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             984..1271
                     /codon_start=1
                     /gene="SEC61B"
                     /product="human Sec61-beta subunit of the translocon in the
                     endoplasmic reticulum"
                     /label=Sec61-beta
                     /translation="MPGPTPSGTNVGSSGRSPSKAVAARAAGSTVRQRKNASCGTRSAG
                     RTTSAGTGGMWRFYTEDSPGLKVGPVPVLVMSLLFIASVFMLHIWGKYTRS"
     CDS             1290..1331
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1338..2294
                     /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"
     primer_bind     complement(2345..2362)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2351..2575
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2708..2727)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2859..2906
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             2914..3936
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2914..3936
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4066..4187
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4103..4122)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4157..4176
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4236..4252)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4236..4252)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4249..4271)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4260..4276
                     /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(4284..4290,4291..4308,4309..4314))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4329..4350
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4467..4484)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4638..5226)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4718..4737)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5397..6188,6189..6257))
                     /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(6258..6362)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag cttatgcctg gtccgacccc cagtggcact aacgtgggat
     1021 cctcagggcg ctctcccagc aaagcagtgg ccgcccgggc ggcgggatcc actgtccggc
     1081 agaggaaaaa tgccagctgt gggacaagga gtgcaggccg cacaacctcg gcaggcaccg
     1141 gggggatgtg gcgattctac acagaagatt cacctgggct caaagttggc cctgttccag
     1201 tattggttat gagtcttctg ttcatcgctt ctgtatttat gttgcacatt tggggcaagt
     1261 acactcgttc gggatccggt accatcgatg gcaagcccat ccccaacccc ctgctgggcc
     1321 tggacagcac cgctagcaaa gacaatactg tgcctctgaa gctgatcgct ctcctggcta
     1381 atggcgagtt ccatagtggc gaacagctgg gagaaaccct gggcatgtcc agggccgcta
     1441 tcaacaagca cattcagact ctgcgcgact ggggcgtgga cgtgttcacc gtgcccggaa
     1501 agggctactc tctgcccgag cctatcccgc tgctgaacgc taaacagatt ctgggacagc
     1561 tggacggcgg gagcgtggca gtcctgcctg tggtcgactc caccaatcag tacctgctgg
     1621 atcgaatcgg cgagctgaag agtggggatg cttgcattgc agaatatcag caggcaggga
     1681 gaggaagcag agggaggaaa tggttctctc cttttggagc taacctgtac ctgagtatgt
     1741 tttggcgcct gaagcgggga ccagcagcaa tcggcctggg cccggtcatc ggaattgtca
     1801 tggcagaagc gctgcgaaag ctgggagcag acaaggtgcg agtcaaatgg cccaatgacc
     1861 tgtatctgca ggatagaaag ctggcaggca tcctggtgga gctggccgga ataacaggcg
     1921 atgctgcaca gatcgtcatt ggcgccggga ttaacgtggc tatgaggcgc gtggaggaaa
     1981 gcgtggtcaa tcagggctgg atcacactgc aggaagcagg gattaacctg gacaggaata
     2041 ctctggccgc tacgctgatc cgagagctgc gggcagccct ggaactgttc gagcaggaag
     2101 gcctggctcc atatctgcca cggtgggaga agctggataa cttcatcaat agacccgtga
     2161 agctgatcat tggggacaaa gagattttcg ggattagccg ggggattgat aaacagggag
     2221 ccctgctgct ggaacaggac ggagttatca aaccctggat gggcggagaa atcagtctgc
     2281 ggtctgccga aaagtaatga gcggccgctc gagtctagag ggcccgttta aacccgctga
     2341 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     2401 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     2461 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     2521 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
     2581 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
     2641 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     2701 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     2761 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     2821 cccaaaaaac ttgattaggg tgatggttca cgtacctaga agttcctatt ccgaagttcc
     2881 tattctctag aaagtatagg aacttccttg gccaaaaagc ctgaactcac cgcgacgtct
     2941 gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag
     3001 ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta
     3061 aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc
     3121 gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc
     3181 atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct
     3241 gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg
     3301 agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc
     3361 atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc
     3421 agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa
     3481 gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc
     3541 ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc
     3601 aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag
     3661 cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt
     3721 cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag
     3781 ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc
     3841 cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac
     3901 cgacgcccca gcactcgtcc gagggcaaag gaatagcacg tactacgaga tttcgattcc
     3961 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     4021 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca
     4081 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     4141 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
     4201 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     4261 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     4321 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     4381 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     4441 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     4501 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     4561 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4621 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4681 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4741 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     4801 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     4861 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     4921 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     4981 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     5041 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     5101 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     5161 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     5221 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     5281 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     5341 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     5401 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     5461 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     5521 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     5581 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     5641 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     5701 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     5761 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     5821 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     5881 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     5941 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     6001 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     6061 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     6121 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     6181 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     6241 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     6301 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     6361 cgcacatttc cccgaaaagt gccacctgac gtc
//
