LOCUS       sequence-342018-        7728 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for MlaE Leu99Bpa mutant for in vivo 
            crosslinking.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7728)
  AUTHORS   Coudray N, Isom GL, MacRae MR, Saiduddin MN, Bhabha G, Ekiert DC
  TITLE     Structure of bacterial phospholipid transporter MlaFEDB with 
            substrate bound.
  JOURNAL   Elife. 2020 Nov 25;9. pii: 62518. doi: 10.7554/eLife.62518.
  PUBMED    33236984
REFERENCE   2  (bases 1 to 7728)
  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: "2020-11-25"; volume: "9. pii: 62518. doi"; pages: " 
            10.7554/eLife.62518"
COMMENT     Sequence Label: pBEL2063
FEATURES             Location/Qualifiers
     source          1..7728
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        93..377
                     /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     300..319
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             1231..1248
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1261..1281
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(3679..3696)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3849..3935
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4027..4054
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        4073..4164
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4165..4233,4234..5025)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4383..4402)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5067..5522
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5154..5173)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5364..5385
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     rep_origin      5633..6220
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6121..6140
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6374..6391
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     6557..6579
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6717..6736
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(join(6916..7728,1..66))
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 acccgccacc agatgggcat taaacgagta tcccggcagc aggggatcat tttgcgcttc
       61 agccatactt ttcatactcc cgccattcag agaagaaacc aattgtccat attgcatcag
      121 acattgccgt cactgcgtct tttactggct cttctcgcta accaaaccgg taaccccgct
      181 tattaaaagc attctgtaac aaagcgggac caaagccatg acaaaaacgc gtaacaaaag
      241 tgtctataat cacggcagaa aagtccacat tgattatttg cacggcgtca cactttgcta
      301 tgccatagca tttttatcca taagattagc ggatcctacc tgacgctttt tatcgcaact
      361 ctctactgtt tctccatacc cgtttttttg ggctagccga agggtgaatt atggagcagt
      421 ctgtggcgaa tttagtcgat atgcgcgatg tcagttttac gcgtggcaat cgctgcatct
      481 tcgataatat ttccctgacc gtgccgcgag ggaagatcac ggcgatcatg gggccatcgg
      541 gcatcggtaa aacgacgcta ctccgtctga ttggcgggca aatcgcacca gatcatggtg
      601 agatcctttt cgatggtgag aatattccgg cgatgtctcg ttcgcgcctg tatacagtgc
      661 gcaaacggat gagcatgtta tttcagtccg gggcgttgtt cactgatatg aacgtatttg
      721 acaacgtcgc ctatccactg cgcgaacata cccaacttcc cgcgccattg ttgcatagta
      781 cggtgatgat gaagctggag gccgtggggc tgcgtggagc ggctaaacta atgccttctg
      841 aactttccgg tgggatggcg cggcgtgcag cgctggcgcg tgcgattgcg ctggagccgg
      901 atctcatcat gtttgatgaa ccttttgttg ggcaagatcc catcaccatg ggcgtactgg
      961 tgaagctgat ttctgagctg aacagcgcgc tgggcgtgac ttgtgtggtg gtttctcacg
     1021 atgtgccgga agtgttaagt attgcggatc acgcctggat cctggcggac aaaaaaattg
     1081 tcgctcatgg cagtgcccag gcgttgcagg cgaatcctga tccgcgcgta cgtcagtttc
     1141 tggacgggat agctgacggg cctgttccgt tccgctatcc tgccggcgat tatcacgctg
     1201 atcttttacc agggagttaa gccactcatg catcaccacc atcaccatca gcatcaacac
     1261 gaaaatctgt attttcaggg gatgctgtta aatgcgctgg cgtcgctcgg acataaaggg
     1321 attaaaaccc tgagaacgtt cgggcgggcc gggttaatgt tattcaatgc gctggtcggc
     1381 aaaccggaat ttcgcaaaca tgcgccgctg ctggtgcgcc agctctataa tgtcggcgtc
     1441 ctgtcgatgc tgattattgt ggtttctggc gtgttcatcg gaatggtgtt ggggctgcaa
     1501 ggttatctgg ttctgaccac ttatagtgcg gaaaccagtc tgggtatgct ggtggcgtta
     1561 tcgctactgc gtgaataggg gccggtggtt gccgcgttgt tgtttgccgg gcgtgctggt
     1621 tcggcgctaa ccgcagaaat cggcctgatg cgcgctacag agcaactctc cagtatggag
     1681 atgatggcgg tggatccgct gcgtcgggtt atttctcccc gtttctgggc tggggttatt
     1741 tcattaccac tgttgacggt tattttcgtc gccgtgggga tctggggcgg atctctggtc
     1801 ggcgtcagtt ggaaaggcat tgatagcggg ttcttctggt cggcaatgca aaatgccgtc
     1861 gactggcgta tggatctggt caactgtctg attaagagcg tggtgttcgc catcacggtg
     1921 acgtggattt cgttgtttaa cggctacgac gccatcccga cgtctgccgg gattagccgg
     1981 gcaaccactc gcaccgttgt ccactcgtct ctggctgttc tggggctgga ttttgtgctg
     2041 accgcattga tgtttgggaa ttgagttcat gcaaacgaaa aaaaatgaaa tttgggtggg
     2101 tatcttttta ttagcagcac tgctggcggc gctgtttgtt tgcctgaagg cggcgaacgt
     2161 gacgtccata cgtactgaac cgacctacac gctttatgcg acgttcgata acattggcgg
     2221 cctgaaagcg cgctctccgg tcagtattgg tggcgttgtt gtgggtcggg tggcggatat
     2281 tacgctggac ccgaaaacct atctgccgcg cgtaacgctg gaaattgaac aacgttataa
     2341 ccacattcca gataccagtt cgctgagcat tcgtacttcc ggcctgctgg gggaacaata
     2401 tctggcatta aacgtcggtt ttgaagaccc ggaactgggg actgctatcc tgaaggatgg
     2461 cgatacaatt caggacacta agtctgcgat ggtgctggaa gatctcattg gtcagttcct
     2521 ttacggtagt aaaggcgatg acaataagaa tagtggcgat gcgccagctg ctgcgccagg
     2581 taataatgaa accactgaac ctgtgggtac aacgaaataa tttcaggaga accgacgcat
     2641 gtttaaacgt ttaatgatgg tcgctttgct ggtgattgca cctctgagtg cggcaaccgc
     2701 ggcagaccag accaatccgt ataagctgat ggacgaggcg gcgcagaaaa cgttcgatcg
     2761 cctgaagaat gagcaaccgc aaattcgggc caacccggat tatctgcgta ccattgttga
     2821 tcaggaactg ctgccatacg tacaggtgaa atacgccggt gcgctggtgc tgggccagta
     2881 ttacaagagt gcgacccctg ctcaacgtga agcctacttt gccgctttcc gtgagtacct
     2941 gaagcaggct tacggtcagg cgctggcgat gtatcacggt caaacctatc agattgcgcc
     3001 agaacagccg ctgggcgata aaaccattgt gcctattcgc gttaccatta ttgacccgaa
     3061 tggccgtccg ccggtgcgtc tggacttcca gtggcgtaaa aactcccaga cgggcaattg
     3121 gcaggcttac gacatgattg ctgaaggcgt cagtatgatc accaccaaac aaaacgagtg
     3181 gggaacgctg ctgcgtacca aaggtatcga cggcctgact gcgcaactga aatcgatttc
     3241 tcaacagaaa atcactctgg aagagaaaaa ataatgagcg agtcactgag ctggatgcag
     3301 acgggtgaca cgctggcgtt atccggagag ctggatcagg acgttttgct accgctttgg
     3361 gaaatgcgtg aggaagcggt gaaggggatt acctgcatcg atcttagccg tgtctcccgc
     3421 gtggatacgg ggggactggc actgctgctc catcttatcg atctggcgaa aaagcagggc
     3481 aacaatgtga cgcttcaggg ggtaaacgac aaagtgtata ccctggcgaa attgtataat
     3541 ttgcctgctg atgttctgcc tcgttaaggt accaaattcc agaaaagaga cgctgaaaag
     3601 cgtctttttt cgttttggtc ctggtctgag gaatggttgt aagcttggct gttttggcgg
     3661 atgagagaag attttcagcc tgatacagat taaatcagaa cgcagaagcg gtctgataaa
     3721 acagaatttg cctggcggca gtagcgcggt ggtcccacct gaccccatgc cgaactcaga
     3781 agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc catgcgagag tagggaactg
     3841 ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
     3901 gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagcggat ttgaacgttg
     3961 cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa
     4021 ttaagcagaa ggccatcctg acggatggcc tttttgcgtt tctacaaact cttttgttta
     4081 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     4141 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     4201 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     4261 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     4321 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     4381 ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc aagagcaact cggtcgccgc
     4441 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     4501 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     4561 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     4621 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     4681 aacgacgagc gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta
     4741 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     4801 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     4861 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     4921 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     4981 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     5041 tactcatata tactttagat tgatttacgc gccctgtagc ggcgcattaa gcgcggcggg
     5101 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     5161 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     5221 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     5281 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     5341 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     5401 tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     5461 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
     5521 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     5581 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     5641 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     5701 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc tcagcagagc
     5761 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     5821 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     5881 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     5941 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     6001 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     6061 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     6121 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     6181 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     6241 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     6301 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     6361 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     6421 tctccttacg catctgtgcg gtatttcaca ccgcataggg tcatggctgc gccccgacac
     6481 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     6541 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     6601 cgcgcgaggc agaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
     6661 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
     6721 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
     6781 cgtccggcgt agaggatctg ctcatgtttg acagcttatc atcgatgcat aatgtgcctg
     6841 tcaaatggac gaagcaggga ttctgcaaac cctatgctac tccgtcaagc cgtcaattgt
     6901 ctgattcgtt accaattatg acaacttgac ggctacatca ttcacttttt cttcacaacc
     6961 ggcacggaac tcgctcgggc tggccccggt gcatttttta aatacccgcg agaaatagag
     7021 ttgatcgtca aaaccaacat tgcgaccgac ggtggcgata ggcatccggg tggtgctcaa
     7081 aagcagcttc gcctggctga tacgttggtc ctcgcgccag cttaagacgc taatccctaa
     7141 ctgctggcgg aaaagatgtg acagacgcga cggcgacaag caaacatgct gtgcgacgct
     7201 ggcgatatca aaattgctgt ctgccaggtg atcgctgatg tactgacaag cctcgcgtac
     7261 ccgattatcc atcggtggat ggagcgactc gttaatcgct tccatgcgcc gcagtaacaa
     7321 ttgctcaagc agatttatcg ccagcagctc cgaatagcgc ccttcccctt gcccggcgtt
     7381 aatgatttgc ccaaacaggt cgctgaaatg cggctggtgc gcttcatccg ggcgaaagaa
     7441 ccccgtattg gcaaatattg acggccagtt aagccattca tgccagtagg cgcgcggacg
     7501 aaagtaaacc cactggtgat accattcgcg agcctccgga tgacgaccgt agtgatgaat
     7561 ctctcctggc gggaacagca aaatatcacc cggtcggcaa acaaattctc gtccctgatt
     7621 tttcaccacc ccctgaccgc gaatggtgag attgagaata taacctttca ttcccagcgg
     7681 tcggtcgata aaaaaatcga gataaccgtt ggcctcaatc ggcgttaa
//
