LOCUS       sequence-330376-        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"
     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"
     protein_bind    586..604
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    607..625
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     635..659
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             750..1037
                     /codon_start=1
                     /gene="SEC61B"
                     /product="human Sec61-beta subunit of the translocon in the
                     endoplasmic reticulum"
                     /label=Sec61-beta
                     /translation="MPGPTPSGTNVGSSGRSPSKAVAARAAGSTVRQRKNASCGTRSAG
                     RTTSAGTGGMWRFYTEDSPGLKVGPVPVLVMSLLFIASVFMLHIWGKYTRS"
     CDS             1056..1097
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1104..2060
                     /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(2111..2128)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2117..2341
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2474..2493)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2625..2672
                     /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             2680..3702
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2680..3702
                     /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    3832..3953
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3869..3888)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3923..3942
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4002..4018)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4002..4018)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4015..4037)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4026..4042
                     /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(4050..4056,4057..4074,4075..4080))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4095..4116
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4233..4250)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4404..4992)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4484..4503)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5163..5954,5955..6023))
                     /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(6024..6128)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6203..6222)
                     /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 agctctccct atcagtgata
      601 gagatctccc tatcagtgat agagatcgtc gacgagctcg tttagtgaac cgtcagatcg
      661 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc
      721 tccggactct agcgtttaaa cttaagctta tgcctggtcc gacccccagt ggcactaacg
      781 tgggatcctc agggcgctct cccagcaaag cagtggccgc ccgggcggcg ggatccactg
      841 tccggcagag gaaaaatgcc agctgtggga caaggagtgc aggccgcaca acctcggcag
      901 gcaccggggg gatgtggcga ttctacacag aagattcacc tgggctcaaa gttggccctg
      961 ttccagtatt ggttatgagt cttctgttca tcgcttctgt atttatgttg cacatttggg
     1021 gcaagtacac tcgttcggga tccggtacca tcgatggcaa gcccatcccc aaccccctgc
     1081 tgggcctgga cagcaccgct agcaaagaca atactgtgcc tctgaagctg atcgctctcc
     1141 tggctaatgg cgagttccat agtggcgaac agctgggaga aaccctgggc atgtccaggg
     1201 ccgctatcaa caagcacatt cagactctgc gcgactgggg cgtggacgtg ttcaccgtgc
     1261 ccggaaaggg ctactctctg cccgagccta tcccgctgct gaacgctaaa cagattctgg
     1321 gacagctgga cggcgggagc gtggcagtcc tgcctgtggt cgactccacc aatcagtacc
     1381 tgctggatcg aatcggcgag ctgaagagtg gggatgcttg cattgcagaa tatcagcagg
     1441 cagggagagg aagcagaggg aggaaatggt tctctccttt tggagctaac ctgtacctga
     1501 gtatgttttg gcgcctgaag cggggaccag cagcaatcgg cctgggcccg gtcatcggaa
     1561 ttgtcatggc agaagcgctg cgaaagctgg gagcagacaa ggtgcgagtc aaatggccca
     1621 atgacctgta tctgcaggat agaaagctgg caggcatcct ggtggagctg gccggaataa
     1681 caggcgatgc tgcacagatc gtcattggcg ccgggattaa cgtggctatg aggcgcgtgg
     1741 aggaaagcgt ggtcaatcag ggctggatca cactgcagga agcagggatt aacctggaca
     1801 ggaatactct ggccgctacg ctgatccgag agctgcgggc agccctggaa ctgttcgagc
     1861 aggaaggcct ggctccatat ctgccacggt gggagaagct ggataacttc atcaatagac
     1921 ccgtgaagct gatcattggg gacaaagaga ttttcgggat tagccggggg attgataaac
     1981 agggagccct gctgctggaa caggacggag ttatcaaacc ctggatgggc ggagaaatca
     2041 gtctgcggtc tgccgaaaag taatgagcgg ccgctcgagt ctagagggcc cgtttaaacc
     2101 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     2161 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     2221 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     2281 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     2341 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc
     2401 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     2461 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     2521 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     2581 ctcgacccca aaaaacttga ttagggtgat ggttcacgta cctagaagtt cctattccga
     2641 agttcctatt ctctagaaag tataggaact tccttggcca aaaagcctga actcaccgcg
     2701 acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg tctccgacct gatgcagctc
     2761 tcggagggcg aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg
     2821 cgggtaaata gctgcgccga tggtttctac aaagatcgtt atgtttatcg gcactttgca
     2881 tcggccgcgc tcccgattcc ggaagtgctt gacattgggg aattcagcga gagcctgacc
     2941 tattgcatct cccgccgtgc acagggtgtc acgttgcaag acctgcctga aaccgaactg
     3001 cccgctgttc tgcagccggt cgcggaggcc atggatgcga tcgctgcggc cgatcttagc
     3061 cagacgagcg ggttcggccc attcggaccg caaggaatcg gtcaatacac tacatggcgt
     3121 gatttcatat gcgcgattgc tgatccccat gtgtatcact ggcaaactgt gatggacgac
     3181 accgtcagtg cgtccgtcgc gcaggctctc gatgagctga tgctttgggc cgaggactgc
     3241 cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca acaatgtcct gacggacaat
     3301 ggccgcataa cagcggtcat tgactggagc gaggcgatgt tcggggattc ccaatacgag
     3361 gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta tggagcagca gacgcgctac
     3421 ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc tccgggcgta tatgctccgc
     3481 attggtcttg accaactcta tcagagcttg gttgacggca atttcgatga tgcagcttgg
     3541 gcgcagggtc gatgcgacgc aatcgtccga tccggagccg ggactgtcgg gcgtacacaa
     3601 atcgcccgca gaagcgcggc cgtctggacc gatggctgtg tagaagtact cgccgatagt
     3661 ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat agcacgtact acgagatttc
     3721 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     3781 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt
     3841 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     3901 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     3961 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg
     4021 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     4081 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     4141 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     4201 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     4261 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     4321 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     4381 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     4441 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     4501 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     4561 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     4621 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     4681 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     4741 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     4801 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     4861 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     4921 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     4981 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     5041 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     5101 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     5161 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     5221 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     5281 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     5341 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     5401 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     5461 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     5521 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     5581 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     5641 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     5701 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     5761 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     5821 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     5881 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     5941 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     6001 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     6061 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     6121 gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg
     6181 atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc
     6241 tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa
     6301 caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc
     6361 tgcttcgcga tgtacgggcc agatatacgc gtt
//
