LOCUS       sequence-385335-        6126 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression/purification of 6xHis-TEV-MlaC(N155A).
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: pBEL2560
FEATURES             Location/Qualifiers
     source          1..6126
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    11..150
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(76..95)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     236..258
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(252..443)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1252..2334)
                     /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     2296..2315
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2335..2412)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2618..2637
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2721..2740
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2721..2739
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2740..2764
                     /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             2813..2830
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2831..2851
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQA"
     primer_bind     complement(3487..3505)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3501..3548
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3585..4040
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3672..3691)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3882..3903
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(4115..4148)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4227..4331
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4332..4400,4401..5192)
                     /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(4550..4569)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5363..5951
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5852..5871
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6105..6122
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
       61 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
      121 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
      181 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
      241 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
      301 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
      361 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
      421 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
      481 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
      541 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
      601 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
      661 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
      721 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
      781 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
      841 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
      901 cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
      961 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     1021 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     1081 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     1141 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     1201 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     1261 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     1321 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     1381 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     1441 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     1501 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
     1561 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     1621 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     1681 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     1741 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     1801 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     1861 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     1921 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     1981 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     2041 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     2101 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     2161 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     2221 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     2281 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     2341 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     2401 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     2461 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     2521 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     2581 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     2641 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
     2701 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
     2761 ttcccctcta gaaataattt tgtttaactt tggcccagga gatatacata tgcatcacca
     2821 ccatcaccat gaaaatctgt attttcaggc agaccagacc aatccgtata agctgatgga
     2881 cgaggcggcg cagaaaacgt tcgatcgcct gaagaatgag caaccgcaaa ttcgggccaa
     2941 cccggattat ctgcgtacca ttgttgatca ggaactgctg ccatacgtac aggtgaaata
     3001 cgccggtgcg ctggtgctgg gccagtatta caagagtgcg acccctgctc aacgtgaagc
     3061 ctactttgcc gctttccgtg agtacctgaa gcaggcttac ggtcaggcgc tggcgatgta
     3121 tcacggtcaa acctatcaga ttgcgccaga acagccgctg ggcgataaaa ccattgtgcc
     3181 tattcgcgtt accattattg acccgaatgg ccgtccgccg gtgcgtctgg acttccagtg
     3241 gcgtaaagcg tcccagacgg gcaattggca ggcttacgac atgattgctg aaggcgtcag
     3301 tatgatcacc accaaacaaa acgagtgggg aacgctgctg cgtaccaaag gtatcgacgg
     3361 cctgactgcg caactgaaat cgatttctca acagaaaatc actctggaag agaaaaaata
     3421 agagagaccg gccggccgat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     3481 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     3541 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg
     3601 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     3661 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     3721 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     3781 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     3841 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     3901 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     3961 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     4021 aatattaacg cttacaattt aggtggcact tttcggggaa cgacgacctg cagccaagct
     4081 agcttggctg gacgtaaact cctcttcaga cctaataact tcgtatagca tacattatac
     4141 gaagttatat taagggttat tgaatatgat cggaattggg ctgcaggaat tcgatagctt
     4201 ggctgcaggt cgacctcgag gatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     4261 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     4321 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     4381 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     4441 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     4501 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     4561 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     4621 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     4681 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     4741 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     4801 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     4861 acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     4921 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     4981 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     5041 agcgtggctc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     5101 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     5161 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     5221 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     5281 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     5341 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     5401 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     5461 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     5521 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     5581 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     5641 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     5701 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     5761 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     5821 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     5881 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     5941 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     6001 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     6061 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     6121 aggaag
//
