LOCUS       sequence-384565-        6126 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression/purification of 6xHis-TEV-MlaC(L76D).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6126)
  AUTHORS   MacRae MR, Puvanendran D, Haase MAB, Coudray N, Kolich L, Lam C, 
            Baek M, Bhabha G, Ekiert DC
  TITLE     Protein-protein interactions in the Mla lipid transport system 
            probed by computational structure prediction and deep mutational 
            scanning.
  JOURNAL   J Biol Chem. 2023 Apr 24:104744. doi: 10.1016/j.jbc.2023.104744.
  PUBMED    37100290
REFERENCE   2  (bases 1 to 6126)
  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: "J Biol 
            Chem. 2023 Apr 24:104744. doi: 10.1016/j.jbc.2023.104744."
COMMENT     Sequence Label: pBEL2559
FEATURES             Location/Qualifiers
     source          1..6126
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      292..880
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     781..800
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1034..1051
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1066..1205
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1131..1150)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1291..1313
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1307..1498)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2307..3389)
                     /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     3351..3370
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3390..3467)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3673..3692
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3776..3795
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3776..3794
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3795..3819
                     /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)."
     CDS             3868..3885
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3886..3906
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQA"
     primer_bind     complement(4542..4560)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4556..4603
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4640..5095
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4727..4746)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4937..4958
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(5170..5203)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        5282..5386
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5387..5455,5456..6126,1..121)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5605..5624)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
       61 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
      121 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
      181 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
      241 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
      301 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
      361 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
      421 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
      481 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
      541 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
      601 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
      661 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
      721 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
      781 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
      841 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
      901 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
      961 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     1021 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     1081 tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc
     1141 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
     1201 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     1261 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     1321 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
     1381 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
     1441 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
     1501 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
     1561 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
     1621 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
     1681 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
     1741 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
     1801 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
     1861 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
     1921 ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg
     1981 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
     2041 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
     2101 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
     2161 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
     2221 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     2281 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     2341 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     2401 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     2461 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     2521 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     2581 accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     2641 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     2701 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     2761 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
     2821 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
     2881 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
     2941 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
     3001 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
     3061 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
     3121 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
     3181 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
     3241 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
     3301 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
     3361 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     3421 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
     3481 ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag
     3541 caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac
     3601 ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg
     3661 atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt
     3721 gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata
     3781 cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt
     3841 aactttggcc caggagatat acatatgcat caccaccatc accatgaaaa tctgtatttt
     3901 caggcagacc agaccaatcc gtataagctg atggacgagg cggcgcagaa aacgttcgat
     3961 cgcctgaaga atgagcaacc gcaaattcgg gccaacccgg attatctgcg taccattgtt
     4021 gatcaggaac tgctgccata cgtacaggtg aaatacgccg gtgcggatgt gctgggccag
     4081 tattacaaga gtgcgacccc tgctcaacgt gaagcctact ttgccgcttt ccgtgagtac
     4141 ctgaagcagg cttacggtca ggcgctggcg atgtatcacg gtcaaaccta tcagattgcg
     4201 ccagaacagc cgctgggcga taaaaccatt gtgcctattc gcgttaccat tattgacccg
     4261 aatggccgtc cgccggtgcg tctggacttc cagtggcgta aaaactccca gacgggcaat
     4321 tggcaggctt acgacatgat tgctgaaggc gtcagtatga tcaccaccaa acaaaacgag
     4381 tggggaacgc tgctgcgtac caaaggtatc gacggcctga ctgcgcaact gaaatcgatt
     4441 tctcaacaga aaatcactct ggaagagaaa aaataagaga gaccggccgg ccgatccggc
     4501 tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc
     4561 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat
     4621 atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     4681 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     4741 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     4801 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     4861 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     4921 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     4981 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     5041 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
     5101 gcacttttcg gggaacgacg acctgcagcc aagctagctt ggctggacgt aaactcctct
     5161 tcagacctaa taacttcgta tagcatacat tatacgaagt tatattaagg gttattgaat
     5221 atgatcggaa ttgggctgca ggaattcgat agcttggctg caggtcgacc tcgaggatgt
     5281 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     5341 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     5401 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     5461 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     5521 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     5581 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     5641 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     5701 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     5761 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     5821 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     5881 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc
     5941 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     6001 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     6061 tggctggttt attgctgata aatctggagc cggtgagcgt ggctctcgcg gtatcattgc
     6121 agcact
//
