LOCUS       sequence-243577-        5540 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express endogenous biotin ligase in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5540)
  AUTHORS   Lomakin YA, Kaminskaya AN, Stepanov AV, Shmidt AA, Gabibov A, 
            Belogurov AA
  TITLE     Probing surface membrane receptors using engineered bacteriophage 
            bioconjugates.
  JOURNAL   Bioconjug Chem. 2019 Apr 25. doi: 10.1021/acs.bioconjchem.9b00218.
  PUBMED    31021608
REFERENCE   2  (bases 1 to 5540)
  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: "Bioconjug 
            Chem. 2019 Apr 25. doi: 10.1021/acs.bioconjchem.9b00218."
COMMENT     Sequence Label: BirA His-tag pLEMO
FEATURES             Location/Qualifiers
     source          1..5540
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             7..969
                     /codon_start=1
                     /gene="birA"
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSSEK"
     CDS             982..999
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1055..1073)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1069..1116
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1337..1996)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1911..1930
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1997..2099)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(2491..2508)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2625..3170)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(2705..2724)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3501..4349)
                     /codon_start=1
                     /gene="E. coli rhaR"
                     /product="transcriptional activator of rhaR and rhaS"
                     /label=rhaR
                     /note="needed for rhamnose-dependent activation of the rhaB
                     promoter"
                     /translation="MAHQLKLLKDDFFASDQQAVAVADRYPQDVFAEHTHDFCELVIVW
                     RGNGLHVLNDRPYRITRGDLFYIHADDKHSYASVNDLVLQNIIYCPERLKLNLDWQGAI
                     PGFNASAGQPHWRLGSMGMAQARQVIGQLEHESSQHVPFANEMAELLFGQLVMLLNRHR
                     YTSDSLPPTSSETLLDKLITRLAASLKSPFALDKFCDEASCSERVLRQQFRQQTGMTIN
                     QYLRQVRVCHAQYLLQHSRLLISDISTECGFEDSNYFSVVFTRETGMTPSQWRHLNSQK
                     D"
     CDS             complement(4423..5259)
                     /codon_start=1
                     /gene="E. coli rhaS"
                     /product="positive regulator of the rhaB promoter
                     "
                     /label=rhaS
                     /note="activated by L-rhamnose"
                     /translation="MTVLHSVDFFPSGNASVAIEPRLPQADFPEHHHDFHEIVIVEHGT
                     GIHVFNGQPYTITGGTVCFVRDHDRHLYEHTDNLCLTNVLYRSPDRFQFLAGLNQLLPQ
                     ELDGQYPSHWRVNHSVLQQVRQLVAQMEQQEGENDLPSTASREILFMQLLLLLRKSSLQ
                     ENLENSASRLNLLLAWLEDHFADEVNWDAVADQFSLSLRTLHRQLKQQTGLTPQRYLNR
                     LRLMKARHLLRHSEASVTDIAYRCGFSDSNHFSTLFRREFNWSPRDIRQGRDGFLQ"
     promoter        5405..5523
                     /label=rhaB promoter
                     /note="promoter of the E. coli rhaBAD operon, conferring 
                     tight induction with L-rhamnose and repression with 
                     D-glucose in the presence of RhaR and RhaS (Giacalone et 
                     al., 2006)"
ORIGIN
        1 gtcgacatga aggataacac cgtgccactg aaattgattg ccctgttagc gaacggtgaa
       61 tttcactctg gcgagcagtt gggtgaaacg ctgggaatga gccgggcggc tattaataaa
      121 cacattcaga cactgcgtga ctggggcgtt gatgtcttta ccgttccggg taaaggatac
      181 agcctgcctg agcctatcca gttacttaat gctaaacaga tattgggtca gctggatggc
      241 ggtagtgtag ccgtgctgcc agtgattgac tccacgaatc agtaccttct tgatcgtatc
      301 ggagagctta aatcgggcga tgcttgcatt gcagaatacc agcaggctgg ccgtggtcgc
      361 cggggtcgga aatggttttc gccttttggc gcaaacttat atttgtcgat gttctggcgt
      421 ctggaacaag gcccggcggc ggcgattggt ttaagtctgg ttatcggtat cgtgatggcg
      481 gaagtattac gcaagctggg tgcagataaa gttcgtgtta aatggcctaa tgacctctat
      541 ctgcaggatc gcaagctggc aggcattctg gtggagctga ctggcaaaac tggcgatgcg
      601 gcgcaaatag tcattggagc cgggatcaac atggcaatgc gccgtgttga agagagtgtc
      661 gttaatcagg ggtggatcac gctgcaggaa gcggggatca atctcgatcg taatacgttg
      721 gcggccatgc taatacgtga attacgtgct gcgttggaac tcttcgaaca agaaggattg
      781 gcaccttatc tgtcgcgctg ggaaaagctg gataatttta ttaatcgccc agtgaaactt
      841 atcattggtg ataaagaaat atttggcatt tcacgcggaa tagacaaaca gggggcttta
      901 ttacttgagc aggatggaat aataaaaccc tggatgggcg gtgaaatatc cctgcgtagt
      961 tcagaaaaaa ctggtctcga gcaccaccac caccaccacg gatccccgcg ccctcatccg
     1021 aaagggcgta ttcatatgcc ctaggctgct gccaccgctg agcaataact agcataaccc
     1081 cttggggcct ctaaacgggt cttgaggggt tttttgctga aacctcaggc atttgagaag
     1141 cacacggtca cactgcttcc ggtagtcaat aaaccggtaa accagcaata gacataagcg
     1201 gctatttaac gaccctgccc tgaaccgacg accgggtcga atttgctttc gaatttctgc
     1261 cattcatccg cttattatca cttattcagg cgtagcaacc aggcgtttaa gggcaccaat
     1321 aactgcctta aaaaaattac gccccgccct gccactcatc gcagtactgt tgtaattcat
     1381 taagcattct gccgacatgg aagccatcac aaacggcatg atgaacctga atcgccagcg
     1441 gcatcagcac cttgtcgcct tgcgtataat atttgcccat agtgaaaacg ggggcgaaga
     1501 agttgtccat attggccacg tttaaatcaa aactggtgaa actcacccag ggattggctg
     1561 agacgaaaaa catattctca ataaaccctt tagggaaata ggccaggttt tcaccgtaac
     1621 acgccacatc ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg tattcactcc
     1681 agagcgatga aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg tgaacactat
     1741 cccatatcac cagctcaccg tctttcattg ccatacggaa ctccggatga gcattcatca
     1801 ggcgggcaag aatgtgaata aaggccggat aaaacttgtg cttatttttc tttacggtct
     1861 ttaaaaaggc cgtaatatcc agctgaacgg tctggttata ggtacattga gcaactgact
     1921 gaaatgcctc aaaatgttct ttacgatgcc attgggatat atcaacggtg gtatatccag
     1981 tgattttttt ctccatttta gcttccttag ctcctgaaaa tctcgataac tcaaaaaata
     2041 cgcccggtag tgatcttatt tcattatggt gaaagttgga acctcttacg tgccgatcaa
     2101 cgtctcattt tcgccaaaag ttggcccagg gcttcccggt atcaacaggg acaccaggat
     2161 ttatttattc tgcgaagtga tcttccgtca caggtattta ttcggcgcaa agtgcgtcgg
     2221 gtgatgctgc caacttactg atttagtgta tgatggtgtt tttgaggtgc tccagtggct
     2281 tctgtttcta tcagctgtcc ctcctgttca gctactgacg gggtggtgcg taacggcaaa
     2341 agcaccgccg gacatcagcg ctagcggagt gtatactggc ttactatgtt ggcactgatg
     2401 agggtgtcag tgaagtgctt catgtggcag gagaaaaaag gctgcaccgg tgcgtcagca
     2461 gaatatgtga tacaggatat attccgcttc ctcgctcact gactcgctac gctcggtcgt
     2521 tcgactgcgg cgagcggaaa tggcttacga acggggcgga gatttcctgg aagatgccag
     2581 gaagatactt aacagggaag tgagagggcc gcggcaaagc cgtttttcca taggctccgc
     2641 ccccctgaca agcatcacga aatctgacgc tcaaatcagt ggtggcgaaa cccgacagga
     2701 ctataaagat accaggcgtt tccccctggc ggctccctcg tgcgctctcc tgttcctgcc
     2761 tttcggttta ccggtgtcat tccgctgtta tggccgcgtt tgtctcattc cacgcctgac
     2821 actcagttcc gggtaggcag ttcgctccaa gctggactgt atgcacgaac cccccgttca
     2881 gtccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg aaagacatgc
     2941 aaaagcacca ctggcagcag ccactggtaa ttgatttaga ggagttagtc ttgaagtcat
     3001 gcgccggtta aggctaaact gaaaggacaa gttttggtga ctgcgctcct ccaagccagt
     3061 tacctcggtt caaagagttg gtagctcaga gaaccttcga aaaaccgccc tgcaaggcgg
     3121 ttttttcgtt ttcagagcaa gagattacgc gcagaccaaa acgatctcaa gaagatcatc
     3181 ttattaatca gataaaatat ttctaggttt cagtgcaatt tatctcttca aatgtagcac
     3241 ctgaagtcag ccccatacga tataagttgt aattctcatg ttagtcatgc cccgcgccca
     3301 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     3361 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     3421 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     3481 tgggcgcccg ataagcttaa ttaatctttc tgcgaattga gatgacgcca ctggctgggc
     3541 gtcatcccgg tttcccgggt aaacaccacc gaaaaatagt tactatcttc aaagccacat
     3601 tcggtcgaaa tatcactgat taacaggcgg ctatgctgga gaagatattg cgcatgacac
     3661 actctgacct gtcgcagata ttgattgatg gtcattccag tctgctggcg aaattgctga
     3721 cgcaaaacgc gctcactgca cgatgcctca tcacaaaatt tatccagcgc aaagggactt
     3781 ttcaggctag ccgccagccg ggtaatcagc ttatccagca acgtttcgct ggatgttggc
     3841 ggcaacgaat cactggtgta acgatggcga ttcagcaaca tcaccaactg cccgaacagc
     3901 aactcagcca tttcgttagc aaacggcaca tgctgactac tttcatgctc aagctgacca
     3961 ataacctgcc gcgcctgcgc catccccatg ctacctaagc gccagtgtgg ttgccctgcg
     4021 ctggcgttaa atcccggaat cgccccctgc cagtcaagat tcagcttcag acgctccggg
     4081 caataaataa tattctgcaa aaccagatcg ttaacggaag cgtaggagtg tttatcatca
     4141 gcatgaatgt aaaagagatc gccacgggta atgcgataag ggcgatcgtt gagtacatgc
     4201 aggccattac cgcgccagac aatcaccagc tcacaaaaat catgtgtatg ttcagcaaag
     4261 acatcttgcg gataacggtc agccacagcg actgcctgct ggtcgctggc aaaaaaatca
     4321 tctttgagaa gttttaactg atgcgccacc gtggctacct cggccagaga acgaagttga
     4381 ttattcgcaa tatggcgtac aaatacgttg agaagattcg cgttattgca gaaagccatc
     4441 ccgtccctgg cgaatatcac gcggtgacca gttaaactct cggcgaaaaa gcgtcgaaaa
     4501 gtggttactg tcgctgaatc cacagcgata ggcgatgtca gtaacgctgg cctcgctgtg
     4561 gcgtagcaga tgtcgggctt tcatcagtcg caggcggttc aggtatcgct gaggcgtcag
     4621 tcccgtttgc tgcttaagct gccgatgtag cgtacgcagt gaaagagaaa attgatccgc
     4681 cacggcatcc caattcacct catcggcaaa atggtcctcc agccaggcca gaagcaagtt
     4741 gagacgtgat gcgctgtttt ccaggttctc ctgcaaactg cttttacgca gcaagagcag
     4801 taattgcata aacaagatct cgcgactggc ggtcgagggt aaatcatttt ccccttcctg
     4861 ctgttccatc tgtgcaacca gctgtcgcac ctgctgcaat acgctgtggt taacgcgcca
     4921 gtgagacgga tactgcccat ccagctcttg tggcagcaac tgattcagcc cggcgagaaa
     4981 ctgaaatcga tccggcgagc gatacagcac attggtcaga cacagattat cggtatgttc
     5041 atacagatgc cgatcatgat cgcgtacgaa acagaccgtg ccaccggtga tggtataggg
     5101 ctgcccatta aacacatgaa tacccgtgcc atgttcgaca atcacaattt catgaaaatc
     5161 atgatgatgt tcaggaaaat ccgcctgcgg gagccggggt tctatcgcca cggacgcgtt
     5221 accagacgga aaaaaatcca cactatgtaa tacggtcata ctggcctcct gatgtcgtca
     5281 acacggcgaa atagtaatca cgaggtcagg ttcttacctt aaattttcga cggaaaacca
     5341 cgtaaaaaac gtcgattttt caagatacag cgtgaatttt caggaaatgc ggtgagcatc
     5401 acatcaccac aattcagcaa attgtgaaca tcatcacgtt catctttccc tggttgccaa
     5461 tggcccattt tcctgtcagt aacgagaagg tcgcgaattc aggcgctttt tagactggtc
     5521 gtaatgaaat tcaggaggtg
//
