LOCUS       sequence-240571-        5917 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses BirA and eCPX-AP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5917)
  AUTHORS   Granhoj J, Dimke H, Svenningsen P
  TITLE     A bacterial display system for effective selection of protein-biotin
            ligase BirA variants with novel peptide specificity.
  JOURNAL   Sci Rep. 2019 Mar 11;9(1):4118. doi: 10.1038/s41598-019-40984-x.
  PUBMED    30858523
REFERENCE   2  (bases 1 to 5917)
  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: "Sci Rep."; 
            date: "2019-03-11"; volume: "9(1):4118. doi"; pages: " 
            10.1038/s41598-019-40984-x"
COMMENT     Sequence Label: pBAD-BirA-eCPX-AP
FEATURES             Location/Qualifiers
     source          1..5917
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..285
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     208..227
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     RBS             312..334
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             346..1308
                     /codon_start=1
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             346..1308
                     /codon_start=1
                     /gene="birA"
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             1309..1326
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1384..1401)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1554..1640
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1732..1759
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1778..1869
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1870..1938,1939..2730)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2088..2107)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2901..3489
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3390..3409
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3548..3595)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     3591..3609
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(3733..3777)
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     protein_bind    4395..4419
                     /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)."
     primer_bind     complement(4419..4438)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4420..4438)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     4577..4594
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4609..4749
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4675..4694)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4835..4857
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5013..5891)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct
       61 tctcgctaac caaaccggta accccgctta ttaaaagcat tctgtaacaa agcgggacca
      121 aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg
      181 attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg
      241 atcctacctg acgcttttta tcgcaactct ctactgtttc tccatacccg tttttttggg
      301 ctagaaataa ttttgtttaa ctttaagaag gagatataca tacccatgaa ggataacacc
      361 gtgccactga aattgattgc cctgttagcg aacggtgaat ttcactctgg cgagcagttg
      421 ggtgaaacgc tgggaatgag ccgggcggct attaataaac acattcagac actgcgtgac
      481 tggggcgttg atgtctttac cgttccgggt aaaggataca gcctgcctga gcctatccag
      541 ttacttaatg ctaaacagat attgggtcag ctggatggcg gtagtgtagc cgtgctgcca
      601 gtgattgact ccacgaatca gtaccttctt gatcgtatcg gagagcttaa atcgggcgat
      661 gcttgcattg cagaatacca gcaggctggc cgtggtcgcc ggggtcggaa atggttttcg
      721 ccttttggcg caaacttata tttgtcgatg ttctggcgtc tggaacaagg cccggcggcg
      781 gcgattggtt taagtctggt tatcggtatc gtgatggcgg aagtattacg caagctgggt
      841 gcagataaag ttcgtgttaa atggcctaat gacctctatc tgcaggatcg caagctggca
      901 ggcattctgg tggagctgac tggcaaaact ggcgatgcgg cgcaaatagt cattggagcc
      961 gggatcaaca tggcaatgcg ccgtgttgaa gagagtgtcg ttaatcaggg gtggatcacg
     1021 ctgcaggaag cggggatcaa tctcgatcgt aatacgttgg cggccatgct aatacgtgaa
     1081 ttacgtgctg cgttggaact cttcgaacaa gaaggattgg caccttatct gtcgcgctgg
     1141 gaaaagctgg ataattttat taatcgccca gtgaaactta tcattggtga taaagaaata
     1201 tttggcattt cacgcggaat agacaaacag ggggctttat tacttgagca ggatggaata
     1261 ataaaaccct ggatgggcgg tgaaatatcc ctgcgtagtg cagaaaaaca tcatcatcat
     1321 catcattgag agtttaaacg gtctccagct tggctgtttt ggcggatgag agaagatttt
     1381 cagcctgata cagattaaat cagaacgcag aagcggtctg ataaaacaga atttgcctgg
     1441 cggcagtagc gcggtggtcc cacctgaccc catgccgaac tcagaagtga aacgccgtag
     1501 cgccgatggt agtgtggggt ctccccatgc gagagtaggg aactgccagg catcaaataa
     1561 aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg
     1621 ctctcctgag taggacaaat ccgccgggag cggatttgaa cgttgcgaag caacggcccg
     1681 gagggtggcg ggcaggacgc ccgccataaa ctgccaggca tcaaattaag cagaaggcca
     1741 tcctgacgga tggccttttt gcgtttctac aaactctttt gtttattttt ctaaatacat
     1801 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     1861 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     1921 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     1981 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     2041 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     2101 gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     2161 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     2221 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     2281 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     2341 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     2401 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     2461 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     2521 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     2581 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     2641 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     2701 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     2761 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     2821 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     2881 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     2941 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     3001 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     3061 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     3121 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     3181 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     3241 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     3301 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     3361 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     3421 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     3481 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tgaaccggat atagttcctc
     3541 ctttcagcaa aaaacccctc aagacccgtt tagaggcccc aaggggttat gctagttatt
     3601 gctcagcggt ggcagcagcc aactcagctt cctttcgggc tttgttagca gccggatcag
     3661 cttgcatgcc tgcaggtcga ctctagagga tccccgggta ccgagctcga attcgtttaa
     3721 acattactta agctcgtgcc actcgatttt ctgtgcttca aaaatatcat tcagaccgcg
     3781 gccgcctgca cgacgtgcac cgcttgcggt acggcttttt tcggtatagg taaagctacc
     3841 aataacaccc agcggactat tatcttcttc gtagcgatat ttcagattaa agccacccat
     3901 tttattcatc tgaccctgtg catcactctg tgcataacca ccggtaacgg tgctggttgc
     3961 acgacggctt ttgctaccaa aacgataacc aacgctcaga atccaggtgc caacatcaac
     4021 gctacgaata cggctctgtt catagctgaa atccagtgca acattttcca tcggattaaa
     4081 ctgcagacct gcaccatagc taaaaccata atcgctggta tcatgtttat aggtcggata
     4141 ttcggtggtc tgaaatttac cataaccaac acccacaaca ccataaatgc ttgcccaatc
     4201 attaatacga tatgccggac cggctgtaat gccgtaatac tgatttttgt tataatcacc
     4261 actctgaccg ctctgaccac cggcaacgct ggtgcctgcg gtaaatgcca gaactgctgc
     4321 cagtgcgctc agacatgcga tttttttcat ggcgatcgct ccttactaaa gttaaagaaa
     4381 attattacta gtggggaatt gttatccgct cacaattccc ctatagtgag tcgtattaat
     4441 ttcgcgggat cgagatccct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     4501 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     4561 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
     4621 ttttctcctt acgcatctgt gcggtatttc ataccgcata tggtgcactc tcagtacaat
     4681 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     4741 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     4801 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     4861 tcaccgtcat caccgaaacg cgcgaggcag cagatcaatt cgcgcgcgaa ggcgaagcgg
     4921 catgcataat gtgcctgtca aatggacgaa gcagggattc tgcaaaccct atgctactcc
     4981 gtcaagccgt caattgtctg attcgttacc aattatgaca acttgacggc tacatcattc
     5041 actttttctt cacaaccggc acggaactcg ctcgggctgg ccccggtgca ttttttaaat
     5101 acccgcgaga aatagagttg atcgtcaaaa ccaacattgc gaccgacggt ggcgataggc
     5161 atccgggtgg tgctcaaaag cagcttcgcc tggctgatac gttggtcctc gcgccagctt
     5221 aagacgctaa tccctaactg ctggcggaaa agatgtgaca gacgcgacgg cgacaagcaa
     5281 acatgctgtg cgacgctggc gatatcaaaa ttgctgtctg ccaggtgatc gctgatgtac
     5341 tgacaagcct cgcgtacccg attatccatc ggtggatgga gcgactcgtt aatcgcttcc
     5401 atgcgccgca gtaacaattg ctcaagcaga tttatcgcca gcagctccga atagcgccct
     5461 tccccttgcc cggcgttaat gatttgccca aacaggtcgc tgaaatgcgg ctggtgcgct
     5521 tcatccgggc gaaagaaccc cgtattggca aatattgacg gccagttaag ccattcatgc
     5581 cagtaggcgc gcggacgaaa gtaaacccac tggtgatacc attcgcgagc ctccggatga
     5641 cgaccgtagt gatgaatctc tcctggcggg aacagcaaaa tatcacccgg tcggcaaaca
     5701 aattctcgtc cctgattttt caccaccccc tgaccgcgaa tggtgagatt gagaatataa
     5761 cctttcattc ccagcggtcg gtcgataaaa aaatcgagat aaccgttggc ctcaatcggc
     5821 gttaaacccg ccaccagatg ggcattaaac gagtatcccg gcagcagggg atcattttgc
     5881 gcttcagcca tacttttcat actcccgcca ttcagag
//
