LOCUS       sequence-344822-        6242 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expression of Methionine Binding Protein (MetQ) with C20A mutation 
            in signal sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6242)
  AUTHORS   Sharaf NG, Shahgholi M, Kim E, Lai JY, VanderVelde DG, Lee AT, Rees 
            DC
  TITLE     Characterization of the ABC methionine transporter from Neisseria 
            meningitidis reveals that lipidated MetQ is required for 
            interaction.
  JOURNAL   Elife. 2021 Aug 19;10. pii: 69742. doi: 10.7554/eLife.69742.
  PUBMED    34409939
REFERENCE   2  (bases 1 to 6242)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Elife."; 
            date: "2021-08-19"; volume: "10. pii: 69742. doi"; pages: " 
            10.7554/eLife.69742"
COMMENT     Sequence Label: NmMetQ C20A
FEATURES             Location/Qualifiers
     source          1..6242
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     141..160
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     244..263
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        244..262
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    263..287
                     /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)."
     RBS             302..324
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1199..1228
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(1287..1305)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1301..1348
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1385..1840
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1472..1491)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1682..1703
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1866..1970
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1971..2039,2040..2831)
                     /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(2189..2208)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3002..3590
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3491..3510
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3744..3761
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3776..3915
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3841..3860)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4001..4023
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4017..4208)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5017..6099)
                     /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     6061..6080
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6100..6177)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
       61 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
      121 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
      181 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga
      241 aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa
      301 ttttgtttaa ctttaagaag gagatataca tatgaaaacc ttcttcaaaa ccctttccgc
      361 cgccgcactc gcgctcatcc tcgccgccgc cggcggtcaa aaagatagcg cgcccgccgc
      421 atccgcttct gccgccgccg acaacggcgc ggcgaaaaaa gaaatcgtct tcggcacgac
      481 cgtcggcgac ttcggcgata tggtcaaaga acaaatccaa gccgagctgg agaaaaaagg
      541 ctacaccgtc aaactggtcg agtttaccga ctatgtgcgc ccgaatctgg cattggctga
      601 gggcgagttg gacatcaacg tcttccaaca caaaccctat cttgacgact tcaaaaaaga
      661 acacaatctg gacatcaccg aagtcttcca agtgccgacc gcgcctttgg gactgtaccc
      721 gggcaagctg aaatcgctgg aagaagtcaa agacggcagc accgtatccg cgcccaacga
      781 cccgtccaac ttcgcccgcg tcttggtgat gctcgacgaa ctgggttgga tcaaactcaa
      841 agacggcatc aatccgctga ccgcatccaa agcggacatt gccgaaaacc tgaaaaacat
      901 caaaatcgtc gagcttgaag ccgcgcaact gccgcgtagc cgcgccgacg tggattttgc
      961 cgtcgtcaac ggcaactacg ccataagcag cggcatgaag ctgaccgaag ccctgttcca
     1021 agaaccgagc tttgcctatg tcaactggtc tgccgtcaaa accgccgaca aagacagcca
     1081 atggcttaaa gacgtaaccg aggcctataa ctccgacgcg ttcaaagcct acgcgcacaa
     1141 acgcttcgag ggctacaaat cccctgccgc atggaatgaa ggcgcagcca aactcgagca
     1201 tcatcatcat catcatcatc atcatcactg actcgaggat ccggctgcta acaaagcccg
     1261 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
     1321 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa
     1381 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     1441 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     1501 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     1561 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     1621 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     1681 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     1741 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     1801 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa
     1861 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     1921 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     1981 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     2041 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     2101 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     2161 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     2221 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     2281 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     2341 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     2401 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     2461 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa
     2521 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     2581 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     2641 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     2701 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     2761 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     2821 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     2881 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     2941 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     3001 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     3061 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     3121 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     3181 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     3241 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     3301 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     3361 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     3421 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     3481 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     3541 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     3601 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     3661 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     3721 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     3781 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag
     3841 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
     3901 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     3961 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     4021 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
     4081 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
     4141 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
     4201 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
     4261 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
     4321 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
     4381 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
     4441 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt
     4501 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag
     4561 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa
     4621 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc
     4681 gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg
     4741 aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc
     4801 gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct
     4861 acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc
     4921 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     4981 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     5041 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     5101 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     5161 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     5221 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     5281 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     5341 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     5401 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     5461 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     5521 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     5581 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     5641 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     5701 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     5761 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     5821 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     5881 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     5941 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     6001 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     6061 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     6121 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     6181 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     6241 gg
//
