LOCUS       sequence-338018-        6440 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express TurboID_18A in Bl21.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6440)
  AUTHORS   Bao H
  TITLE     Developing Nanodisc-ID for label-free characterizations of membrane 
            proteins.
  JOURNAL   Commun Biol. 2021 Apr 30;4(1):514. doi: 10.1038/s42003-021-02043-y.
  PUBMED    33931748
REFERENCE   2  (bases 1 to 6440)
  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: "Commun 
            Biol."; date: "2021-04-30"; volume: "4(1):514. doi"; pages: " 
            10.1038/s42003-021-02043-y"
COMMENT     Sequence Label: pet21_TurboID_18A
FEATURES             Location/Qualifiers
     source          1..6440
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             146..175
                     /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="ASMTGGQQMG"
     CDS             191..1150
                     /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             1172..1189
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1242..1260)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1256..1303
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1340..1795
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1427..1446)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1637..1658
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1821..1925
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1926..1994,1995..2786)
                     /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      2957..3545
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3446..3465
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3699..3716
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3731..3870
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3796..3815)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3956..3978
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3972..4163)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4972..6054)
                     /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"
     primer_bind     6016..6035
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6055..6132)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6338..6357
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat ggactggttc aaagctttct acgacaaagt
      121 tgctgaaaaa ttcaaagaag ctttcgctag catgactggt ggacagcaaa tgggtcgcgg
      181 atccgaattc atgaaagaca atactgtgcc tctgaagctg atcgctctcc tggctaatgg
      241 cgagttccat agtggcgaac agctgggaga aaccctgggc atgtccaggg ccgctatcaa
      301 caagcacatt cagactctgc gcgactgggg cgtggacgtg ttcaccgtgc ccggaaaggg
      361 ctactctctg cccgagccta tcccgctgct gaacgctaaa cagattctgg gacagctgga
      421 cggcgggagc gtggcagtcc tgcctgtggt cgactccacc aatcagtacc tgctggatcg
      481 aatcggcgag ctgaagagtg gggatgcttg cattgcagaa tatcagcagg cagggagagg
      541 aagcagaggg aggaaatggt tctctccttt tggagctaac ctgtacctga gtatgttttg
      601 gcgcctgaag cggggaccag cagcaatcgg cctgggcccg gtcatcggaa ttgtcatggc
      661 agaagcgctg cgaaagctgg gagcagacaa ggtgcgagtc aaatggccca atgacctgta
      721 tctgcaggat agaaagctgg caggcatcct ggtggagctg gccggaataa caggcgatgc
      781 tgcacagatc gtcattggcg ccgggattaa cgtggctatg aggcgcgtgg aggaaagcgt
      841 ggtcaatcag ggctggatca cactgcagga agcagggatt aacctggaca ggaatactct
      901 ggccgctacg ctgatccgag agctgcgggc agccctggaa ctgttcgagc aggaaggcct
      961 ggctccatat ctgccacggt gggagaagct ggataacttc atcaatagac ccgtgaagct
     1021 gatcattggg gacaaagaga ttttcgggat tagccggggg attgataaac agggagccct
     1081 gctgctggaa caggacggag ttatcaaacc ctggatgggc ggagaaatca gtctgcggtc
     1141 tgccgaaaag aagcttgcgg ccgcactcga gcaccaccac caccaccact gagatccggc
     1201 tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc
     1261 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat
     1321 atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     1381 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     1441 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     1501 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     1561 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     1621 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     1681 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     1741 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
     1801 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     1861 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     1921 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     1981 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     2041 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     2101 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     2161 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     2221 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     2281 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     2341 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     2401 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     2461 cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     2521 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     2581 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     2641 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     2701 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     2761 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     2821 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     2881 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     2941 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     3001 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     3061 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     3121 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     3181 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     3241 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     3301 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     3361 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     3421 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     3481 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     3541 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     3601 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     3661 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     3721 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     3781 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     3841 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     3901 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     3961 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     4021 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     4081 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     4141 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     4201 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     4261 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     4321 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     4381 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     4441 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     4501 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     4561 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     4621 cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     4681 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     4741 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     4801 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     4861 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     4921 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     4981 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     5041 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     5101 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     5161 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     5221 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
     5281 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     5341 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     5401 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     5461 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     5521 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     5581 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     5641 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     5701 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     5761 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     5821 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     5881 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     5941 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     6001 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     6061 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     6121 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     6181 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     6241 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     6301 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     6361 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
     6421 agatctcgat cccgcgaaat
//
