LOCUS       sequence-300459-        5242 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 5242)
  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 5242)
  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..5242
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(9..23,24..29,30..46,47..53)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    29..45
                     /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    61..77
                     /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             96..107
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             501..1466
                     /codon_start=1
                     /gene="birA"
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             501..1463
                     /codon_start=1
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     terminator      1486..1580
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(1628..1647)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1639..2454
                     /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             1639..2454
                     /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(1712..1731)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      2673..2759
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2852..3934)
                     /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     3896..3915
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3935..4012)
                     /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(4096..4118)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4204..4344
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4259..4278
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4359..4376)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4530..5118)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4610..4629)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5209..5227
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgagaaatc ataaaaaatt tatttgcttt gtgagcggat aacaattata atagattcaa
       61 ttgtgagcgg ataacaattt cacacagaat tcattaaaga ggagaaatta accatgagca
      121 agcatcacca tcattcaggc catcaccata ccggacacca ccatcattca ggcagtcatc
      181 accataccgg cggatcatcc ggaggtatga gctcaggatc taacggatct acgggttctg
      241 gtactggtaa tagcgggacc ggaaccatga ttaaagtaaa gactctgacc ggcaaggaaa
      301 ttgagatcga catcgaaccg accgacacca tcgatcgtat caaagaacgt gtggaggaaa
      361 aagaaggcat tcctccggtt cagcagcgtc tcatttacgc gggtaaacag cttgcagatg
      421 acaaaaccgc caaagattac aatatcgagg gcggtagcgt actgcacctt gttctggccc
      481 tgcgtggtgg cgccggtacc atgaaggata acaccgtgcc actgaaattg attgccctgt
      541 tagcgaacgg tgaatttcac tctggcgagc agttgggtga aacgctggga atgagccggg
      601 cggctattaa taaacacatt cagacactgc gtgactgggg cgttgatgtc tttaccgttc
      661 cgggtaaagg atacagcctg cctgagccta tccagttact taatgctaaa cagatattgg
      721 gtcagctgga tggcggtagt gtagccgtgc tgccagtgat tgactccacg aatcagtacc
      781 ttcttgatcg tatcggagag cttaaatcgg gcgatgcttg cattgcagaa taccagcagg
      841 ctggccgtgg tcgccggggt cggaaatggt tttcgccttt tggcgcaaac ttatatttgt
      901 cgatgttctg gcgtctggaa caaggcccgg cggcggcgat tggtttaagt ctggttatcg
      961 gtatcgtgat ggcggaagta ttacgcaagc tgggtgcaga taaagttcgt gttaaatggc
     1021 ctaatgacct ctatctgcag gatcgcaagc tggcaggcat tctggtggag ctgactggca
     1081 aaactggcga tgcggcgcaa atagtcattg gagccgggat caacatggca atgcgccgtg
     1141 ttgaagagag tgtcgttaat caggggtgga tcacgctgca ggaagcgggg atcaatctcg
     1201 atcgtaatac gttggcggcc atgctaatac gtgaattacg tgctgcgttg gaactcttcg
     1261 aacaagaagg attggcacct tatctgtcgc gctgggaaaa gctggataat tttattaatc
     1321 gcccagtgaa acttatcatt ggtgataaag aaatatttgg catttcacgc ggaatagaca
     1381 aacagggggc tttattactt gagcaggatg gaataataaa accctggatg ggcggtgaaa
     1441 tatccctgcg tagtgccgaa aaataagctt aattagctga gcttggactc ctgttgatag
     1501 atccagtaat gacctcagaa ctccatctgg atttgttcag aacgctcggt tgccgccggg
     1561 cgttttttat tggtgagaat ccaagctagc catgaaaata aactgtctgc ttacataaac
     1621 agtaatacaa ggggtgttat gagccatatt caacgggaaa cgtcttgctc taggccgcga
     1681 ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg
     1741 caatcaggtg cgacaatcta tcgattgtat gggaagcccg atgcgccaga gttgtttctg
     1801 aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg
     1861 ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgct
     1921 tggttactca cgactgcgat ccccggcaaa acagcattcc aggtattaga agaatatcct
     1981 gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt
     2041 cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca
     2101 cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct
     2161 gttgaacaag tctggaaaga aatgcacaaa cttttgccat tctcaccgga ttcagtcgtc
     2221 actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt
     2281 attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac
     2341 tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat
     2401 aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaagaatta
     2461 attcatgggc aaatattata cgcaaggcga caaggtgctg atgccgctgg cgattcaggt
     2521 tcatcatgcc gtttgtgatg gcttccatgt cggcagaatg cttaatgaat tacaacagta
     2581 ctgcgatgag tggcagggcg gggcgtaatt tttttaaggc agttattggt gcccttaaac
     2641 gcctggggta atgactctct agcttgaggc atcaaataaa acgaaaggct cagtcgaaag
     2701 actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc
     2761 cgccctctag attacgtgca gtcgatgata agctgtcaaa catgagaatt gtgcctaatg
     2821 agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     2881 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     2941 gggccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
     3001 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
     3061 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
     3121 ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
     3181 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
     3241 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
     3301 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
     3361 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
     3421 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
     3481 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
     3541 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
     3601 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
     3661 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
     3721 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
     3781 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
     3841 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
     3901 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     3961 ccgggcgcta tcatgccata ccgcgaaagg ttttgcacca ttcgatggtg tcggaatttc
     4021 gggcagcgtt gggtcctggc cacgggtgcg catgatctag agctgcctcg cgcgtttcgg
     4081 tgatgacggt gaaaacctct gacacatgca gctcccggca tccgcttaca gacaagctgt
     4141 gaccgtctcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg
     4201 gggcgcagcc atgacccagt cacgtagcga tagcggagtg tatactggct taactatgcg
     4261 gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc
     4321 gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc
     4381 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     4441 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     4501 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4561 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4621 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     4681 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     4741 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     4801 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     4861 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     4921 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
     4981 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     5041 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     5101 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     5161 aacgaaaact cacgttaagg gattttggtc atgacattaa cctataaaaa taggcgtatc
     5221 acgaggccct ttcgtcttca cc
//
