miniTurbo-His6_pET21a

LOCUS       miniTurbo-His6_pET21a                6200 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  expresses His6-tagged miniTurbo in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    miniTurbo-His6_pET21a
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6200)
  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 6200)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..6200
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     48..67
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     151..170
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        151..169
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    170..194
                     /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             209..231
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             239..271
                     /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             290..1060
                     /codon_start=1
                     /product="N-terminally truncated mutant of the E. coli 
                     biotin ligase BioID engineered for improved catalytic 
                     efficiency (Branon et al., 2018)"
                     /label=miniTurbo
                     /translation="IPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACI
                     AEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLGADK
                     VRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQ
                     EAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGDKEIF
                     GISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK"
     CDS             1082..1099
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1152..1170)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1166..1213
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1250..1705
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1337..1356)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1547..1568
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1731..1835
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1836..2696
                     /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"
     primer_bind     complement(2054..2073)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2867..3455
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3356..3375
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3609..3626
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3641..3780
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3706..3725)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3866..3888
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3882..4073)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4882..5964)
                     /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     5926..5945
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5965..6042)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg
       61 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
      121 tagaggatcg agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag
      181 cggataacaa ttcccctcta gaaataattt tgtttaactt taagaaggag atatacatat
      241 ggctagcatg actggtggac agcaaatggg tcgcggatcc gaattcatga tcccgctgct
      301 gaacgctaaa cagattctgg gacagctgga cggcgggagc gtggcagtcc tgcctgtggt
      361 cgactccacc aatcagtacc tgctggatcg aatcggcgag ctgaagagtg gggatgcttg
      421 cattgcagaa tatcagcagg cagggagagg aagcagaggg aggaaatggt tctctccttt
      481 tggagctaac ctgtacctga gtatgttttg gcgcctgaag cggggaccag cagcaatcgg
      541 cctgggcccg gtcatcggaa ttgtcatggc agaagcgctg cgaaagctgg gagcagacaa
      601 ggtgcgagtc aaatggccca atgacctgta tctgcaggat agaaagctgg caggcatcct
      661 ggtggagctg gccggaataa caggcgatgc tgcacagatc gtcattggcg ccgggattaa
      721 cgtggctatg aggcgcgtgg aggaaagcgt ggtcaatcag ggctggatca cactgcagga
      781 agcagggatt aacctggaca ggaatactct ggccgctatg ctgatccgag agctgcgggc
      841 agccctggaa ctgttcgagc aggaaggcct ggctccatat ctgtcacggt gggagaagct
      901 ggataacttc atcaatagac ccgtgaagct gatcattggg gacaaagaga ttttcgggat
      961 tagccggggg attgataaac agggagccct gctgctggaa caggacggag ttatcaaacc
     1021 ctggatgggc ggagaaatca gtctgcggtc tgccgaaaag aagcttgcgg ccgcactcga
     1081 gcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt
     1141 ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
     1201 gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt
     1261 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     1321 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     1381 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     1441 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     1501 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     1561 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     1621 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     1681 aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc
     1741 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     1801 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     1861 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     1921 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     1981 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     2041 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     2101 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     2161 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     2221 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     2281 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     2341 aagccatacc aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc
     2401 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     2461 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     2521 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     2581 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     2641 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     2701 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     2761 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     2821 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     2881 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     2941 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     3001 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     3061 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     3121 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     3181 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     3241 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     3301 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     3361 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     3421 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     3481 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     3541 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     3601 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
     3661 ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga
     3721 tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg
     3781 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     3841 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     3901 tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca
     3961 cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc
     4021 tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc
     4081 tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg
     4141 ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa
     4201 caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc
     4261 ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc
     4321 cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa
     4381 ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac
     4441 gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc
     4501 cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata
     4561 atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg
     4621 gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag
     4681 cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata
     4741 aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg
     4801 actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa
     4861 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4921 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
     4981 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
     5041 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
     5101 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
     5161 gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
     5221 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
     5281 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
     5341 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
     5401 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
     5461 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
     5521 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
     5581 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
     5641 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
     5701 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
     5761 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
     5821 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
     5881 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
     5941 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
     6001 tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc
     6061 ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg
     6121 ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc
     6181 ctgccaccat acccacgccg
//