LOCUS       sequence-447907-        5702 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BioID protein purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5702)
  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 5702)
  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: pNHA6
FEATURES             Location/Qualifiers
     source          1..5702
                     /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             join(151..498,499..501,502..1110)
                     /codon_start=1
                     /product="promiscuous R118G mutant of the E. coli biotin 
                     protein ligase (Cronan, 2005)"
                     /label=BirA(R118G)
                     /note="also known as BirA* or BioID"
                     /translation="KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDW
                     GVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGD
                     ACIAEYQQAGRGGRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRKL
                     GADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQGW
                     ITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGD
                     KEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     terminator      1159..1253
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1297..1956
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1363..1382)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2021..2107
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2200..3282)
                     /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     3244..3263
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3283..3360)
                     /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(3444..3466)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3552..3692
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3607..3626
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3707..3724)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3878..4466)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3958..3977)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4637..5428,5429..5497))
                     /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"
     primer_bind     5260..5279
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5498..5602)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5670..5688
                     /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 aaggacaaca ccgtgcccct gaagctgatc
      181 gccctgctgg ccaacggcga gttccactct ggcgagcagc tgggagagac cctgggaatg
      241 agcagagccg ccatcaacaa gcacatccag acactgagag actggggagt ggacgtgttc
      301 accgtgcctg gcaagggcta cagcctgcct gagcctatcc agctgctgaa cgccaagcag
      361 atcctgggac agctggatgg cggaagcgtg gccgtgctgc ctgtgatcga ctccaccaat
      421 cagtacctgc tggacagaat cggagagctg aagtccggcg acgcctgcat cgccgagtac
      481 cagcaggctg gcagaggagg cagaggacgg aagtggttca gcccattcgg agccaacctg
      541 tacctgtcca tgttctggag actggagcag ggacctgctg ctgccatcgg actgagtctg
      601 gtgatcggaa tcgtgatggc cgaggtgctg agaaagctgg gagccgacaa ggtgagagtg
      661 aagtggccta atgacctgta cctccaggac cgcaagctgg ctggcatcct ggtggagctg
      721 acaggcaaga caggcgatgc cgctcagatc gtgatcggag ccggaatcaa catggccatg
      781 agaagagtgg aggagagcgt ggtgaaccag ggctggatca ccctgcagga ggctggcatc
      841 aacctggacc ggaacaccct ggccgccatg ctgatcagag agctgagagc cgctctggag
      901 ctgttcgagc aggagggact ggctccttac ctgagcagat gggagaagct ggacaacttc
      961 atcaacagac ctgtgaagct gatcatcggc gacaaggaaa tcttcggcat ctccagagga
     1021 atcgacaagc agggagctct gctgctggag caggacggaa tcatcaagcc ctggatgggc
     1081 ggagaaatct ccctgagaag cgcagagaag taaggtaccc cgggtcgacc tgcagccaag
     1141 cttaattagc tgagcttgga ctcctgttga tagatccagt aatgacctca gaactccatc
     1201 tggatttgtt cagaacgctc ggttgccgcc gggcgttttt tattggtgag aatccaagct
     1261 agcttggcga gattttcagg agctaaggaa gctaaaatgg agaaaaaaat cactggatat
     1321 accaccgttg atatatccca atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt
     1381 gctcaatgta cctataacca gaccgttcag ctggatatta cggccttttt aaagaccgta
     1441 aagaaaaata agcacaagtt ttatccggcc tttattcaca ttcttgcccg cctgatgaat
     1501 gctcatccgg aatttcgtat ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt
     1561 cacccttgtt acaccgtttt ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa
     1621 taccacgacg atttccggca gtttctacac atatattcgc aagatgtggc gtgttacggt
     1681 gaaaacctgg cctatttccc taaagggttt attgagaata tgtttttcgt ctcagccaat
     1741 ccctgggtga gtttcaccag ttttgattta aacgtggcca atatggacaa cttcttcgcc
     1801 cccgttttca ccatgggcaa atattatacg caaggcgaca aggtgctgat gccgctggcg
     1861 attcaggttc atcatgccgt ttgtgatggc ttccatgtcg gcagaatgct taatgaatta
     1921 caacagtact gcgatgagtg gcagggcggg gcgtaatttt tttaaggcag ttattggtgc
     1981 ccttaaacgc ctggggtaat gactctctag cttgaggcat caaataaaac gaaaggctca
     2041 gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag
     2101 gacaaatccg ccctctagat tacgtgcagt cgatgataag ctgtcaaaca tgagaattgt
     2161 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     2221 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     2281 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
     2341 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
     2401 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
     2461 gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg
     2521 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
     2581 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
     2641 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
     2701 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
     2761 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
     2821 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
     2881 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
     2941 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
     3001 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
     3061 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
     3121 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     3181 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     3241 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     3301 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcaccatt cgatggtgtc
     3361 ggaatttcgg gcagcgttgg gtcctggcca cgggtgcgca tgatctagag ctgcctcgcg
     3421 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct
     3481 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
     3541 gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta tactggctta
     3601 actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc
     3661 acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact
     3721 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     3781 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     3841 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     3901 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     3961 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     4021 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     4081 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     4141 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     4201 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     4261 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     4321 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     4381 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     4441 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     4501 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     4561 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     4621 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     4681 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     4741 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     4801 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     4861 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     4921 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     4981 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     5041 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     5101 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     5161 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     5221 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     5281 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     5341 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     5401 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     5461 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     5521 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     5581 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     5641 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtcttc
     5701 ac
//
