LOCUS       sequence-320146-        7728 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for MlaE Val77Bpa 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: pBEL2060
FEATURES             Location/Qualifiers
     source          1..7728
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        10..294
                     /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     217..236
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             1148..1165
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1178..1198
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(3596..3613)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3766..3852
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3944..3971
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3990..4081
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4082..4150,4151..4942)
                     /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(4300..4319)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4984..5439
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5071..5090)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5281..5302
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     rep_origin      5550..6137
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6038..6057
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6291..6308
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     6474..6496
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6634..6653
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(6833..7711)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 cattcagaga agaaaccaat tgtccatatt gcatcagaca ttgccgtcac tgcgtctttt
       61 actggctctt ctcgctaacc aaaccggtaa ccccgcttat taaaagcatt ctgtaacaaa
      121 gcgggaccaa agccatgaca aaaacgcgta acaaaagtgt ctataatcac ggcagaaaag
      181 tccacattga ttatttgcac ggcgtcacac tttgctatgc catagcattt ttatccataa
      241 gattagcgga tcctacctga cgctttttat cgcaactctc tactgtttct ccatacccgt
      301 ttttttgggc tagccgaagg gtgaattatg gagcagtctg tggcgaattt agtcgatatg
      361 cgcgatgtca gttttacgcg tggcaatcgc tgcatcttcg ataatatttc cctgaccgtg
      421 ccgcgaggga agatcacggc gatcatgggg ccatcgggca tcggtaaaac gacgctactc
      481 cgtctgattg gcgggcaaat cgcaccagat catggtgaga tccttttcga tggtgagaat
      541 attccggcga tgtctcgttc gcgcctgtat acagtgcgca aacggatgag catgttattt
      601 cagtccgggg cgttgttcac tgatatgaac gtatttgaca acgtcgccta tccactgcgc
      661 gaacataccc aacttcccgc gccattgttg catagtacgg tgatgatgaa gctggaggcc
      721 gtggggctgc gtggagcggc taaactaatg ccttctgaac tttccggtgg gatggcgcgg
      781 cgtgcagcgc tggcgcgtgc gattgcgctg gagccggatc tcatcatgtt tgatgaacct
      841 tttgttgggc aagatcccat caccatgggc gtactggtga agctgatttc tgagctgaac
      901 agcgcgctgg gcgtgacttg tgtggtggtt tctcacgatg tgccggaagt gttaagtatt
      961 gcggatcacg cctggatcct ggcggacaaa aaaattgtcg ctcatggcag tgcccaggcg
     1021 ttgcaggcga atcctgatcc gcgcgtacgt cagtttctgg acgggatagc tgacgggcct
     1081 gttccgttcc gctatcctgc cggcgattat cacgctgatc ttttaccagg gagttaagcc
     1141 actcatgcat caccaccatc accatcagca tcaacacgaa aatctgtatt ttcaggggat
     1201 gctgttaaat gcgctggcgt cgctcggaca taaagggatt aaaaccctga gaacgttcgg
     1261 gcgggccggg ttaatgttat tcaatgcgct ggtcggcaaa ccggaatttc gcaaacatgc
     1321 gccgctgctg gtgcgccagc tctataatgt cggcgtcctg tcgatgctga ttattgtggt
     1381 ttctggcgtg ttcatcggaa tggtgttggg gctgcaaggt tatctgtagc tgaccactta
     1441 tagtgcggaa accagtctgg gtatgctggt ggcgttatcg ctactgcgtg aactggggcc
     1501 ggtggttgcc gcgttgttgt ttgccgggcg tgctggttcg gcgctaaccg cagaaatcgg
     1561 cctgatgcgc gctacagagc aactctccag tatggagatg atggcggtgg atccgctgcg
     1621 tcgggttatt tctccccgtt tctgggctgg ggttatttca ttaccactgt tgacggttat
     1681 tttcgtcgcc gtggggatct ggggcggatc tctggtcggc gtcagttgga aaggcattga
     1741 tagcgggttc ttctggtcgg caatgcaaaa tgccgtcgac tggcgtatgg atctggtcaa
     1801 ctgtctgatt aagagcgtgg tgttcgccat cacggtgacg tggatttcgt tgtttaacgg
     1861 ctacgacgcc atcccgacgt ctgccgggat tagccgggca accactcgca ccgttgtcca
     1921 ctcgtctctg gctgttctgg ggctggattt tgtgctgacc gcattgatgt ttgggaattg
     1981 agttcatgca aacgaaaaaa aatgaaattt gggtgggtat ctttttatta gcagcactgc
     2041 tggcggcgct gtttgtttgc ctgaaggcgg cgaacgtgac gtccatacgt actgaaccga
     2101 cctacacgct ttatgcgacg ttcgataaca ttggcggcct gaaagcgcgc tctccggtca
     2161 gtattggtgg cgttgttgtg ggtcgggtgg cggatattac gctggacccg aaaacctatc
     2221 tgccgcgcgt aacgctggaa attgaacaac gttataacca cattccagat accagttcgc
     2281 tgagcattcg tacttccggc ctgctggggg aacaatatct ggcattaaac gtcggttttg
     2341 aagacccgga actggggact gctatcctga aggatggcga tacaattcag gacactaagt
     2401 ctgcgatggt gctggaagat ctcattggtc agttccttta cggtagtaaa ggcgatgaca
     2461 ataagaatag tggcgatgcg ccagctgctg cgccaggtaa taatgaaacc actgaacctg
     2521 tgggtacaac gaaataattt caggagaacc gacgcatgtt taaacgttta atgatggtcg
     2581 ctttgctggt gattgcacct ctgagtgcgg caaccgcggc agaccagacc aatccgtata
     2641 agctgatgga cgaggcggcg cagaaaacgt tcgatcgcct gaagaatgag caaccgcaaa
     2701 ttcgggccaa cccggattat ctgcgtacca ttgttgatca ggaactgctg ccatacgtac
     2761 aggtgaaata cgccggtgcg ctggtgctgg gccagtatta caagagtgcg acccctgctc
     2821 aacgtgaagc ctactttgcc gctttccgtg agtacctgaa gcaggcttac ggtcaggcgc
     2881 tggcgatgta tcacggtcaa acctatcaga ttgcgccaga acagccgctg ggcgataaaa
     2941 ccattgtgcc tattcgcgtt accattattg acccgaatgg ccgtccgccg gtgcgtctgg
     3001 acttccagtg gcgtaaaaac tcccagacgg gcaattggca ggcttacgac atgattgctg
     3061 aaggcgtcag tatgatcacc accaaacaaa acgagtgggg aacgctgctg cgtaccaaag
     3121 gtatcgacgg cctgactgcg caactgaaat cgatttctca acagaaaatc actctggaag
     3181 agaaaaaata atgagcgagt cactgagctg gatgcagacg ggtgacacgc tggcgttatc
     3241 cggagagctg gatcaggacg ttttgctacc gctttgggaa atgcgtgagg aagcggtgaa
     3301 ggggattacc tgcatcgatc ttagccgtgt ctcccgcgtg gatacggggg gactggcact
     3361 gctgctccat cttatcgatc tggcgaaaaa gcagggcaac aatgtgacgc ttcagggggt
     3421 aaacgacaaa gtgtataccc tggcgaaatt gtataatttg cctgctgatg ttctgcctcg
     3481 ttaaggtacc aaattccaga aaagagacgc tgaaaagcgt cttttttcgt tttggtcctg
     3541 gtctgaggaa tggttgtaag cttggctgtt ttggcggatg agagaagatt ttcagcctga
     3601 tacagattaa atcagaacgc agaagcggtc tgataaaaca gaatttgcct ggcggcagta
     3661 gcgcggtggt cccacctgac cccatgccga actcagaagt gaaacgccgt agcgccgatg
     3721 gtagtgtggg gtctccccat gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag
     3781 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg
     3841 agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg
     3901 cgggcaggac gcccgccata aactgccagg catcaaatta agcagaaggc catcctgacg
     3961 gatggccttt ttgcgtttct acaaactctt ttgtttattt ttctaaatac attcaaatat
     4021 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     4081 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     4141 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     4201 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     4261 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     4321 ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     4381 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     4441 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     4501 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     4561 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     4621 gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     4681 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     4741 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     4801 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     4861 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     4921 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     4981 tttacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     5041 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     5101 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     5161 ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg
     5221 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     5281 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcgggctat tcttttgatt
     5341 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     5401 ttaacgcgaa ttttaacaaa atattaacgt ttacaattta aaaggatcta ggtgaagatc
     5461 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     5521 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     5581 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     5641 ccaactcttt ttccgaaggt aactggctca gcagagcgca gataccaaat actgtccttc
     5701 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     5761 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     5821 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     5881 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     5941 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     6001 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     6061 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     6121 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     6181 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     6241 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     6301 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     6361 tttcacaccg catagggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     6421 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     6481 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcaga aggagatggc
     6541 gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat
     6601 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc
     6661 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatctgctc
     6721 atgtttgaca gcttatcatc gatgcataat gtgcctgtca aatggacgaa gcagggattc
     6781 tgcaaaccct atgctactcc gtcaagccgt caattgtctg attcgttacc aattatgaca
     6841 acttgacggc tacatcattc actttttctt cacaaccggc acggaactcg ctcgggctgg
     6901 ccccggtgca ttttttaaat acccgcgaga aatagagttg atcgtcaaaa ccaacattgc
     6961 gaccgacggt ggcgataggc atccgggtgg tgctcaaaag cagcttcgcc tggctgatac
     7021 gttggtcctc gcgccagctt aagacgctaa tccctaactg ctggcggaaa agatgtgaca
     7081 gacgcgacgg cgacaagcaa acatgctgtg cgacgctggc gatatcaaaa ttgctgtctg
     7141 ccaggtgatc gctgatgtac tgacaagcct cgcgtacccg attatccatc ggtggatgga
     7201 gcgactcgtt aatcgcttcc atgcgccgca gtaacaattg ctcaagcaga tttatcgcca
     7261 gcagctccga atagcgccct tccccttgcc cggcgttaat gatttgccca aacaggtcgc
     7321 tgaaatgcgg ctggtgcgct tcatccgggc gaaagaaccc cgtattggca aatattgacg
     7381 gccagttaag ccattcatgc cagtaggcgc gcggacgaaa gtaaacccac tggtgatacc
     7441 attcgcgagc ctccggatga cgaccgtagt gatgaatctc tcctggcggg aacagcaaaa
     7501 tatcacccgg tcggcaaaca aattctcgtc cctgattttt caccaccccc tgaccgcgaa
     7561 tggtgagatt gagaatataa cctttcattc ccagcggtcg gtcgataaaa aaatcgagat
     7621 aaccgttggc ctcaatcggc gttaaacccg ccaccagatg ggcattaaac gagtatcccg
     7681 gcagcagggg atcattttgc gcttcagcca tacttttcat actcccgc
//
