LOCUS       sequence-447906-        5699 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TurboID protein purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5699)
  AUTHORS   Haidar-Ahmad N, Tomaro K, Lavallee-Adam M, Campbell-Valois F-X
  TITLE     The promiscuous biotin ligase TurboID reveals the proxisome of the 
            T3SS chaperone IpgC in Shigella flexneri.
  JOURNAL   mSphere. 2024 Nov 21;9(11):e0055324. doi: 10.1128/msphere.00553-24. 
            Epub 2024 Oct 31.
  PUBMED    39480076
REFERENCE   2  (bases 1 to 5699)
  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: "mSphere."; 
            date: "2024-11-21"; volume: "9(11):e0055324. doi"; pages: " 
            10.1128/msphere.00553-24. Epub 2024 Oct 31"
COMMENT     Sequence Label: pNHA5
FEATURES             Location/Qualifiers
     source          1..5699
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..24,25..30,31..47,48..54)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    30..46
                     /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)."
     protein_bind    62..78
                     /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             97..108
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             127..144
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             151..1107
                     /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"
     terminator      1156..1250
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1294..1953
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1360..1379)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2018..2104
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2197..3279)
                     /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     3241..3260
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3280..3357)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     primer_bind     complement(3441..3463)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3549..3689
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3604..3623
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3704..3721)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3875..4463)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3955..3974)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4634..5425,5426..5494))
                     /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(5495..5599)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5667..5685
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca
       61 attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aactatgaga
      121 ggatcgcatc accatcacca tcacggatcc aaagacaata ctgtgcctct gaagctgatc
      181 gctctcctgg ctaatggcga gttccatagt ggcgaacagc tgggagaaac cctgggcatg
      241 tccagggccg ctatcaacaa gcacattcag actctgcgcg actggggcgt ggacgtgttc
      301 accgtgcccg gaaagggcta ctctctgccc gagcctatcc cgctgctgaa cgctaaacag
      361 attctgggac agctggacgg cgggagcgtg gcagtcctgc ctgtggtcga ctccaccaat
      421 cagtacctgc tggatcgaat cggcgagctg aagagtgggg atgcttgcat tgcagaatat
      481 cagcaggcag ggagaggaag cagagggagg aaatggttct ctccttttgg agctaacctg
      541 tacctgagta tgttttggcg cctgaagcgg ggaccagcag caatcggcct gggcccggtc
      601 atcggaattg tcatggcaga agcgctgcga aagctgggag cagacaaggt gcgagtcaaa
      661 tggcccaatg acctgtatct gcaggataga aagctggcag gcatcctggt ggagctggcc
      721 ggaataacag gcgatgctgc acagatcgtc attggcgccg ggattaacgt ggctatgagg
      781 cgcgtggagg aaagcgtggt caatcagggc tggatcacac tgcaggaagc agggattaac
      841 ctggacagga atactctggc cgctacgctg atccgagagc tgcgggcagc cctggaactg
      901 ttcgagcagg aaggcctggc tccatatctg ccacggtggg agaagctgga taacttcatc
      961 aatagacccg tgaagctgat cattggggac aaagagattt tcgggattag ccgggggatt
     1021 gataaacagg gagccctgct gctggaacag gacggagtta tcaaaccctg gatgggcgga
     1081 gaaatcagtc tgcggtctgc cgaaaagtaa ggtaccccgg gtcgacctgc agccaagctt
     1141 aattagctga gcttggactc ctgttgatag atccagtaat gacctcagaa ctccatctgg
     1201 atttgttcag aacgctcggt tgccgccggg cgttttttat tggtgagaat ccaagctagc
     1261 ttggcgagat tttcaggagc taaggaagct aaaatggaga aaaaaatcac tggatatacc
     1321 accgttgata tatcccaatg gcatcgtaaa gaacattttg aggcatttca gtcagttgct
     1381 caatgtacct ataaccagac cgttcagctg gatattacgg cctttttaaa gaccgtaaag
     1441 aaaaataagc acaagtttta tccggccttt attcacattc ttgcccgcct gatgaatgct
     1501 catccggaat ttcgtatggc aatgaaagac ggtgagctgg tgatatggga tagtgttcac
     1561 ccttgttaca ccgttttcca tgagcaaact gaaacgtttt catcgctctg gagtgaatac
     1621 cacgacgatt tccggcagtt tctacacata tattcgcaag atgtggcgtg ttacggtgaa
     1681 aacctggcct atttccctaa agggtttatt gagaatatgt ttttcgtctc agccaatccc
     1741 tgggtgagtt tcaccagttt tgatttaaac gtggccaata tggacaactt cttcgccccc
     1801 gttttcacca tgggcaaata ttatacgcaa ggcgacaagg tgctgatgcc gctggcgatt
     1861 caggttcatc atgccgtttg tgatggcttc catgtcggca gaatgcttaa tgaattacaa
     1921 cagtactgcg atgagtggca gggcggggcg taattttttt aaggcagtta ttggtgccct
     1981 taaacgcctg gggtaatgac tctctagctt gaggcatcaa ataaaacgaa aggctcagtc
     2041 gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac
     2101 aaatccgccc tctagattac gtgcagtcga tgataagctg tcaaacatga gaattgtgcc
     2161 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     2221 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     2281 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     2341 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     2401 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     2461 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     2521 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     2581 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     2641 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     2701 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     2761 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     2821 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     2881 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     2941 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     3001 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     3061 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     3121 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     3181 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     3241 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     3301 ctcttccggg cgctatcatg ccataccgcg aaaggttttg caccattcga tggtgtcgga
     3361 atttcgggca gcgttgggtc ctggccacgg gtgcgcatga tctagagctg cctcgcgcgt
     3421 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt
     3481 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg
     3541 tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac tggcttaact
     3601 atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca
     3661 gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc
     3721 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     3781 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     3841 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     3901 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     3961 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     4021 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     4081 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     4141 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     4201 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     4261 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     4321 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     4381 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     4441 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     4501 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     4561 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     4621 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     4681 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     4741 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     4801 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     4861 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     4921 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     4981 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     5041 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     5101 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     5161 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     5221 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     5281 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     5341 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     5401 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     5461 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     5521 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     5581 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca
     5641 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtcttcac
//
