LOCUS       sequence-386937-        6337 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cryo-EM of 6xHis-Foldon-(Gly-Ser-Linker)-MlaC.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6337)
  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 6337)
  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: pBEL2319
FEATURES             Location/Qualifiers
     source          1..6337
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(25..44)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     185..207
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(201..392)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1201..2283)
                     /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     2245..2264
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2284..2361)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2567..2586
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2670..2689
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2670..2688
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2689..2713
                     /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             2770..2787
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3647..3665)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3661..3708
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3745..4200
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3832..3851)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4042..4063
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(4275..4308)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4387..4491
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4492..4560,4561..5352)
                     /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(4710..4729)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5523..6111
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6012..6031
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6265..6282
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(6297..6337,1..99)
                     /label=bom
                     /note="basis of mobility region from pBR322"
ORIGIN
        1 cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
       61 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc
      121 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
      181 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
      241 ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc
      301 gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc
      361 atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg
      421 ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat
      481 gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg
      541 cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt
      601 gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc
      661 gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt
      721 gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat
      781 tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc
      841 acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa
      901 cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc
      961 gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc
     1021 cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg
     1081 gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag
     1141 ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc
     1201 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     1261 cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga
     1321 gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc
     1381 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata
     1441 acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag
     1501 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat
     1561 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc
     1621 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg
     1681 ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat
     1741 ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga
     1801 gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt
     1861 agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag
     1921 cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct
     1981 tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc
     2041 cgcgacaatt tgcgacggcg cgtgcagggc cagactggac gtggcaacgc caatcagcaa
     2101 cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat
     2161 cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg
     2221 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt
     2281 cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt
     2341 tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta
     2401 ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat
     2461 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga
     2521 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga
     2581 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt
     2641 agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc
     2701 ggataacaat tcccctctag aaataatttt gtttaacttt ggcccaggcg gccaggaggg
     2761 tctcacatgc atcaccacca tcaccatcca ggctccggct acatccccga ggccccgcgc
     2821 gacggccagg cctacgtgcg caaggacggc gagtgggtgc tgctgtccac cttcctgagt
     2881 agtggaggcg ggtctggtgg cggctccggc ggaggtagta gtagtggagg cgggtctggt
     2941 ggcggctccg gcggaggtag tagtagtgga ggcgggtctg gtggcggctc cggcggaggt
     3001 agtgcagacc agaccaatcc gtataagctg atggacgagg cggcgcagaa aacgttcgat
     3061 cgcctgaaga atgagcaacc gcaaattcgg gccaacccgg attatctgcg taccattgtt
     3121 gatcaggaac tgctgccata cgtacaggtg aaatacgccg gtgcgctggt gctgggccag
     3181 tattacaaga gtgcgacccc tgctcaacgt gaagcctact ttgccgcttt ccgtgagtac
     3241 ctgaagcagg cttacggtca ggcgctggcg atgtatcacg gtcaaaccta tcagattgcg
     3301 ccagaacagc cgctgggcga taaaaccatt gtgcctattc gcgttaccat tattgacccg
     3361 aatggccgtc cgccggtgcg tctggacttc cagtggcgta aaaactccca gacgggcaat
     3421 tggcaggctt acgacatgat tgctgaaggc gtcagtatga tcaccaccaa acaaaacgag
     3481 tggggaacgc tgctgcgtac caaaggtatc gacggcctga ctgcgcaact gaaatcgatt
     3541 tctcaacaga aaatcactct ggaagagaaa aaataagaga gaccggccag gccggccgat
     3601 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     3661 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     3721 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     3781 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     3841 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     3901 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     3961 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     4021 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     4081 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     4141 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     4201 aggtggcact tttcggggaa cgacgacctg cagccaagct agcttggctg gacgtaaact
     4261 cctcttcaga cctaataact tcgtatagca tacattatac gaagttatat taagggttat
     4321 tgaatatgat cggaattggg ctgcaggaat tcgatagctt ggctgcaggt cgacctcgag
     4381 gatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     4441 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     4501 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     4561 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     4621 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     4681 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     4741 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     4801 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     4861 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     4921 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     4981 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca
     5041 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     5101 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     5161 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggctc tcgcggtatc
     5221 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     5281 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     5341 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     5401 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     5461 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     5521 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     5581 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     5641 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     5701 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     5761 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     5821 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     5881 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     5941 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     6001 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     6061 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     6121 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     6181 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     6241 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     6301 atgcggtatt ttctccttac gcatctgtgc ggtattt
//
