LOCUS       sequence-383005-        6354 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression/purification of 6xHis-TEV-sfGFP-MlaA (227-251; L245N).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6354)
  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 6354)
  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: pBEL2754
FEATURES             Location/Qualifiers
     source          1..6354
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      12..467
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(99..118)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     309..330
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(542..575)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        654..758
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(759..827,828..1619)
                     /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(977..996)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1790..2378
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2279..2298
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2532..2549
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2564..2703
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2629..2648)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2789..2811
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2805..2996)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3805..4887)
                     /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     4849..4868
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4888..4965)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5171..5190
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5274..5293
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5274..5292
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5293..5317
                     /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             5366..5383
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5384..5404
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             5414..6124
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     CDS             5414..6124
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(5441..5469)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     6064..6084
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(6268..6286)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6282..6329
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
      361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      421 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
      481 cggggaacga cgacctgcag ccaagctagc ttggctggac gtaaactcct cttcagacct
      541 aataacttcg tatagcatac attatacgaa gttatattaa gggttattga atatgatcgg
      601 aattgggctg caggaattcg atagcttggc tgcaggtcga cctcgaggat gtgcgcggaa
      661 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
      721 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
      781 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
      841 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
      901 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
      961 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     1021 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     1081 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     1141 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     1201 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     1261 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt
     1321 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     1381 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     1441 ttattgctga taaatctgga gccggtgagc gtggctctcg cggtatcatt gcagcactgg
     1501 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     1561 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     1621 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     1681 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     1741 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     1801 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     1861 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     1921 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     1981 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     2041 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     2101 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     2161 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     2221 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     2281 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     2341 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     2401 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     2461 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     2521 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     2581 tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc
     2641 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct
     2701 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     2761 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     2821 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat
     2881 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat
     2941 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat
     3001 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg
     3061 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt
     3121 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag
     3181 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag
     3241 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg
     3301 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     3361 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
     3421 agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg
     3481 ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg
     3541 agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga
     3601 aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg
     3661 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag
     3721 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
     3781 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     3841 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     3901 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca cggcctggcc
     3961 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     4021 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     4081 cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
     4141 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
     4201 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
     4261 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     4321 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     4381 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
     4441 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
     4501 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
     4561 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
     4621 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     4681 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     4741 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     4801 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     4861 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     4921 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
     4981 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
     5041 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
     5101 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
     5161 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
     5221 tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg
     5281 actcactata ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa
     5341 ctttggccca ggagatatac atatgcatca ccaccatcac catgaaaatc tgtattttca
     5401 gggggagaga ccaagcaaag gagaagaact tttcactgga gttgtcccaa ttcttgttga
     5461 attagatggt gatgttaatg ggcacaaatt ttctgtccgt ggagagggtg aaggtgatgc
     5521 tacaaacgga aaactcaccc ttaaatttat ttgcactact ggaaaactac ctgttccgtg
     5581 gccaacactt gtcactactc tgacctatgg tgttcaatgc ttttcccgtt atccggatca
     5641 catgaaacgg catgactttt tcaagagtgc catgcccgaa ggttatgtac aggaacgcac
     5701 tatatctttc aaagatgacg ggacctacaa gacgcgtgct gaagtcaagt ttgaaggtga
     5761 tacccttgtt aatcgtatcg agttaaaggg tattgatttt aaagaagatg gaaacattct
     5821 tggacacaaa ctcgagtaca actttaactc acacaatgta tacatcacgg cagacaaaca
     5881 aaagaatgga atcaaagcta acttcaaaat tcgccacaac gttgaagatg gttccgttca
     5941 actagcagac cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga
     6001 caaccattac ctgtcgacac aatctgtcct ttcgaaagat cccaacgaaa agcgtgacca
     6061 catggtcctt cttgagtttg taactgctgc tgggattaca catggcatgg atgagctcta
     6121 caaaggcggc gaactcaaac cgcaggaaaa tccgaacgca caagcgattc aggatgataa
     6181 caaagatatt gattctgaat aagagagacc ggccggccga tccggctgct aacaaagccc
     6241 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     6301 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggat
//
