LOCUS       sequence-205302-        6386 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses His6-tagged TurboID in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6386)
  AUTHORS   Branon TC, Bosch JA, Sanchez AD, Udeshi ND, Svinkina T, Carr SA, 
            Feldman JL, Perrimon N, Ting AY
  TITLE     Efficient proximity labeling in living cells and organisms with 
            TurboID.
  JOURNAL   Nat Biotechnol. 2018 Aug 20. pii: nbt.4201. doi: 10.1038/nbt.4201.
  PUBMED    30125270
REFERENCE   2  (bases 1 to 6386)
  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 
            Biotechnol. 2018 Aug 20. pii: nbt.4201. doi: 10.1038/nbt.4201."
COMMENT     Sequence Label: TurboID-His6_pET21a
FEATURES             Location/Qualifiers
     source          1..6386
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     940..959
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(979..1056)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1262..1281
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1365..1384
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1365..1383
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1384..1408
                     /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)."
     RBS             1423..1445
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1453..1485
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             1501..2460
                     /codon_start=1
                     /product="mutant of the E. coli biotin ligase BioID 
                     engineered for improved catalytic efficiency (Branon et 
                     al., 2018)"
                     /label=TurboID
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKL
                     GADKVRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGW
                     ITLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGD
                     KEIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK"
     CDS             2482..2499
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2552..2570)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2566..2613
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2650..3105
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2737..2756)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2947..2968
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3131..3235
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3236..3304,3305..4096)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      4267..4855
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4756..4775
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5009..5026
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5041..5180
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5106..5125)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5266..5288
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5282..5473)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6282..6386,1..978))
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
       61 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
      121 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc
      181 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
      241 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
      301 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
      361 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
      421 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
      481 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
      541 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
      601 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
      661 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
      721 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
      781 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
      841 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
      901 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
      961 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     1021 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat
     1081 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg
     1141 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     1201 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
     1261 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca
     1321 cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac gactcactat
     1381 aggggaattg tgagcggata acaattcccc tctagaaata attttgttta actttaagaa
     1441 ggagatatac atatggctag catgactggt ggacagcaaa tgggtcgcgg atccgaattc
     1501 atgaaagaca atactgtgcc tctgaagctg atcgctctcc tggctaatgg cgagttccat
     1561 agtggcgaac agctgggaga aaccctgggc atgtccaggg ccgctatcaa caagcacatt
     1621 cagactctgc gcgactgggg cgtggacgtg ttcaccgtgc ccggaaaggg ctactctctg
     1681 cccgagccta tcccgctgct gaacgctaaa cagattctgg gacagctgga cggcgggagc
     1741 gtggcagtcc tgcctgtggt cgactccacc aatcagtacc tgctggatcg aatcggcgag
     1801 ctgaagagtg gggatgcttg cattgcagaa tatcagcagg cagggagagg aagcagaggg
     1861 aggaaatggt tctctccttt tggagctaac ctgtacctga gtatgttttg gcgcctgaag
     1921 cggggaccag cagcaatcgg cctgggcccg gtcatcggaa ttgtcatggc agaagcgctg
     1981 cgaaagctgg gagcagacaa ggtgcgagtc aaatggccca atgacctgta tctgcaggat
     2041 agaaagctgg caggcatcct ggtggagctg gccggaataa caggcgatgc tgcacagatc
     2101 gtcattggcg ccgggattaa cgtggctatg aggcgcgtgg aggaaagcgt ggtcaatcag
     2161 ggctggatca cactgcagga agcagggatt aacctggaca ggaatactct ggccgctacg
     2221 ctgatccgag agctgcgggc agccctggaa ctgttcgagc aggaaggcct ggctccatat
     2281 ctgccacggt gggagaagct ggataacttc atcaatagac ccgtgaagct gatcattggg
     2341 gacaaagaga ttttcgggat tagccggggg attgataaac agggagccct gctgctggaa
     2401 caggacggag ttatcaaacc ctggatgggc ggagaaatca gtctgcggtc tgccgaaaag
     2461 aagcttgcgg ccgcactcga gcaccaccac caccaccact gagatccggc tgctaacaaa
     2521 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     2581 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg
     2641 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     2701 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     2761 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     2821 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     2881 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     2941 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     3001 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     3061 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
     3121 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     3181 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     3241 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     3301 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     3361 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     3421 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     3481 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     3541 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     3601 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     3661 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     3721 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc
     3781 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     3841 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     3901 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     3961 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     4021 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     4081 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     4141 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     4201 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     4261 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     4321 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     4381 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     4441 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     4501 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     4561 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     4621 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     4681 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     4741 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     4801 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     4861 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     4921 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     4981 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     5041 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
     5101 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     5161 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     5221 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     5281 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
     5341 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
     5401 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
     5461 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
     5521 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
     5581 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
     5641 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
     5701 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
     5761 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
     5821 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
     5881 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg
     5941 gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
     6001 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
     6061 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct
     6121 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc
     6181 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     6241 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     6301 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     6361 tgcgtattgg gcgccagggt ggtttt
//
