LOCUS       sequence-207074-        3142 bp    DNA     linear   UNA 14-APR-2025
DEFINITION  Bacterial expression of biotin ligase (BirA) with an N-terminal myc 
            epitope.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 3142)
  AUTHORS   Buser DP, Schleicher KD, Prescianotto-Baschong C, Spiess M
  TITLE     A versatile nanobody-based toolkit to analyze retrograde transport 
            from the cell surface.
  JOURNAL   Proc Natl Acad Sci U S A. 2018 Jul 3;115(27):E6227-E6236. doi: 
            10.1073/pnas.1801865115. Epub 2018 Jun 18.
  PUBMED    29915061
REFERENCE   2  (bases 1 to 3142)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1073/pnas.1801865115"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2018-07-3- 3"; volume: "115"; issue: "27"; pages: 
            "E6227-E6236"
COMMENT     Sequence Label: pET21d-myc-BirA
FEATURES             Location/Qualifiers
     source          1..3142
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             complement(404..1486)
                     /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     1448..1467
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1487..1564)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1770..1789
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1873..1892
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1873..1891
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1892..1916
                     /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             1931..1953
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1969..1998
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1999..2964
                     /codon_start=1
                     /gene="birA"
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             1999..2961
                     /codon_start=1
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             2986..3003
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3056..3074)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3070..3117
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
       61 tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
      121 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
      181 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
      241 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
      301 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
      361 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
      421 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
      481 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
      541 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
      601 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
      661 tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg
      721 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
      781 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
      841 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
      901 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
      961 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
     1021 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
     1081 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     1141 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     1201 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     1261 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     1321 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     1381 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     1441 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     1501 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
     1561 tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt
     1621 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc
     1681 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc
     1741 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
     1801 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg
     1861 atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac aattcccctc
     1921 tagaaataat tttgtttaac tttaagaagg agatatacca tggctagcga acagaagctc
     1981 atctccgaag aggatctcat gaaggataac accgtgccac tgaaattgat tgccctgtta
     2041 gcgaacggtg aatttcactc tggcgagcag ttgggtgaaa cgctgggaat gagccgggcg
     2101 gctattaata aacacattca gacactgcgt gactggggcg ttgatgtctt taccgttccg
     2161 ggtaaaggat acagcctgcc tgagcctatc cagttactta atgctaaaca gatattgggt
     2221 cagctggatg gcggtagtgt agccgtgctg ccagtgattg actccacgaa tcagtacctt
     2281 cttgatcgta tcggagagct taaatcgggc gatgcttgca ttgcagaata ccagcaggct
     2341 ggccgtggtc gccggggtcg gaaatggttt tcgccttttg gcgcaaactt atatttgtcg
     2401 atgttctggc gtctggaaca aggcccggcg gcggcgattg gtttaagtct ggttatcggt
     2461 atcgtgatgg cggaagtatt acgcaagctg ggtgcagata aagttcgtgt taaatggcct
     2521 aatgacctct atctgcagga tcgcaagctg gcaggcattc tggtggagct gactggcaaa
     2581 actggcgatg cggcgcaaat agtcattgga gccgggatca acatggcaat gcgccgtgtt
     2641 gaagagagtg tcgttaatca ggggtggatc acgctgcagg aagcggggat caatctcgat
     2701 cgtaatacgt tggcggccat gctaatacgt gaattacgtg ctgcgttgga actcttcgaa
     2761 caagaaggat tggcacctta tctgtcgcgc tgggaaaagc tggataattt tattaatcgc
     2821 ccagtgaaac ttatcattgg tgataaagaa atatttggca tttcacgcgg aatagacaaa
     2881 cagggggctt tattacttga gcaggatgga ataataaaac cctggatggg cggtgaaata
     2941 tccctgcgta gtgcagaaaa atgaaagctt gcggccgcac tcgagcacca ccaccaccac
     3001 cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     3061 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     3121 aaaggaggaa ctatatccgg at
//
