LOCUS       sequence-291950-        5243 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Biotin ligase BirA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5243)
  AUTHORS   Pleiner T, Bates M, Trakhanov S, Lee CT, Schliep JE, Chug H, Bohning
            M, Stark H, Urlaub H, Gorlich D
  TITLE     Nanobodies: site-specific labeling for super-resolution imaging, 
            rapid epitope-mapping and native protein complex isolation.
  JOURNAL   Elife. 2015 Dec 3;4:e11349. doi: 10.7554/eLife.11349.
  PUBMED    26633879
REFERENCE   2  (bases 1 to 5243)
  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: "Elife."; 
            date: "2015-12-3"; volume: "4:e11349. doi"; pages: " 
            10.7554/eLife.11349"
COMMENT     Sequence Label: pTP264
FEATURES             Location/Qualifiers
     source          1..5243
                     /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             502..1467
                     /codon_start=1
                     /gene="birA"
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             502..1464
                     /codon_start=1
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     terminator      1487..1581
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(1629..1648)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1640..2455
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             1640..2455
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1713..1732)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      2674..2760
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2854..3936)
                     /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     3898..3917
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3937..4014)
                     /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(4098..4120)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4206..4346
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4261..4280
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4361..4378)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4532..5120)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4612..4631)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5211..5229
                     /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 aaccatgagc
      121 aagcatcacc atcattcagg ccatcaccat accggacacc accatcattc aggcagtcat
      181 caccataccg gcggatcatc cggaggtatg agctcaggat ctaacggatc tacgggttct
      241 ggtactggta atagcgggac cggaaccatg attaaagtaa agactctgac cggcaaggaa
      301 attgagatcg acatcgaacc gaccgacacc atcgatcgta tcaaagaacg tgtggaggaa
      361 aaagaaggca ttcctccggt tcagcagcgt ctcatttacg cgggtaaaca gcttgcagat
      421 gacaaaaccg ccaaagatta caatatcgag ggcggtagcg tactgcacct tgttctggcc
      481 ctgcgtggtg gcgccggtac catgaaggat aacaccgtgc cactgaaatt gattgccctg
      541 ttagcgaacg gtgaatttca ctctggcgag cagttgggtg aaacgctggg aatgagccgg
      601 gcggctatta ataaacacat tcagacactg cgtgactggg gcgttgatgt ctttaccgtt
      661 ccgggtaaag gatacagcct gcctgagcct atccagttac ttaatgctaa acagatattg
      721 ggtcagctgg atggcggtag tgtagccgtg ctgccagtga ttgactccac gaatcagtac
      781 cttcttgatc gtatcggaga gcttaaatcg ggcgatgctt gcattgcaga ataccagcag
      841 gctggccgtg gtcgccgggg tcggaaatgg ttttcgcctt ttggcgcaaa cttatatttg
      901 tcgatgttct ggcgtctgga acaaggcccg gcggcggcga ttggtttaag tctggttatc
      961 ggtatcgtga tggcggaagt attacgcaag ctgggtgcag ataaagttcg tgttaaatgg
     1021 cctaatgacc tctatctgca ggatcgcaag ctggcaggca ttctggtgga gctgactggc
     1081 aaaactggcg atgcggcgca aatagtcatt ggagccggga tcaacatggc aatgcgccgt
     1141 gttgaagaga gtgtcgttaa tcaggggtgg atcacgctgc aggaagcggg gatcaatctc
     1201 gatcgtaata cgttggcggc catgctaata cgtgaattac gtgctgcgtt ggaactcttc
     1261 gaacaagaag gattggcacc ttatctgtcg cgctgggaaa agctggataa ttttattaat
     1321 cgcccagtga aacttatcat tggtgataaa gaaatatttg gcatttcacg cggaatagac
     1381 aaacaggggg ctttattact tgagcaggat ggaataataa aaccctggat gggcggtgaa
     1441 atatccctgc gtagtgccga aaaataagct taattagctg agcttggact cctgttgata
     1501 gatccagtaa tgacctcaga actccatctg gatttgttca gaacgctcgg ttgccgccgg
     1561 gcgtttttta ttggtgagaa tccaagctag ccatgaaaat aaactgtctg cttacataaa
     1621 cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcttgct ctaggccgcg
     1681 attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg
     1741 gcaatcaggt gcgacaatct atcgattgta tgggaagccc gatgcgccag agttgtttct
     1801 gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg
     1861 gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc
     1921 ttggttactc acgactgcga tccccggcaa aacagcattc caggtattag aagaatatcc
     1981 tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat
     2041 tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc
     2101 acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc
     2161 tgttgaacaa gtctggaaag aaatgcacaa acttttgcca ttctcaccgg attcagtcgt
     2221 cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg
     2281 tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa
     2341 ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga
     2401 taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaagaatt
     2461 aattcatggg caaatattat acgcaaggcg acaaggtgct gatgccgctg gcgattcagg
     2521 ttcatcatgc cgtttgtgat ggcttccatg tcggcagaat gcttaatgaa ttacaacagt
     2581 actgcgatga gtggcagggc ggggcgtaat ttttttaagg cagttattgg tgcccttaaa
     2641 cgcctggggt aatgactctc tagcttgagg catcaaataa aacgaaaggc tcagtcgaaa
     2701 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat
     2761 ccgccgctct agattacgtg cagtcgatga taagctgtca aacatgagaa ttgtgcctaa
     2821 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     2881 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     2941 gggggccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     3001 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     3061 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     3121 tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     3181 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     3241 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     3301 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     3361 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     3421 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     3481 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     3541 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     3601 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     3661 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     3721 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     3781 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     3841 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     3901 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     3961 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcac cattcgatgg tgtcggaatt
     4021 tcgggcagcg ttgggtcctg gccacgggtg cgcatgatct agagctgcct cgcgcgtttc
     4081 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     4141 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     4201 cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg
     4261 cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat
     4321 gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc
     4381 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     4441 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     4501 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     4561 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     4621 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     4681 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     4741 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     4801 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     4861 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     4921 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat
     4981 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     5041 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     5101 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     5161 ggaacgaaaa ctcacgttaa gggattttgg tcatgacatt aacctataaa aataggcgta
     5221 tcacgaggcc ctttcgtctt cac
//
