LOCUS       sequence-228534-        6245 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N229A Mutation in Methionine Binding Protein (MetQ) used to solve 
            crystal structure.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6245)
  AUTHORS   Nguyen PT, Lai JY, Lee AT, Kaiser JT, Rees DC
  TITLE     Noncanonical role for the binding protein in substrate uptake by the
            MetNI methionine ATP Binding Cassette (ABC) transporter.
  JOURNAL   Proc Natl Acad Sci U S A. 2018 Oct 23. pii: 1811003115. doi: 
            10.1073/pnas.1811003115.
  PUBMED    30352853
REFERENCE   2  (bases 1 to 6245)
  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: "Proc Natl 
            Acad Sci U S A. 2018 Oct 23. pii: 1811003115. doi: 
            10.1073/pnas.1811003115."
COMMENT     Sequence Label: N229A Lo-MetQ
FEATURES             Location/Qualifiers
     source          1..6245
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     sig_peptide     1..66
                     /codon_start=1
                     /gene="pelB (fragment)"
                     /product="leader peptide for secretion"
                     /label=pelB signal sequence
                     /translation="MKYLLPTAAAGLLLLAAQPAMA"
     CDS             73..102
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             118..132
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             889..906
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(959..977)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      973..1020
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1057..1512
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1144..1163)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1354..1375
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1538..1642
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1643..1711,1712..2503)
                     /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(1861..1880)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2674..3262
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3163..3182
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3416..3433
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3448..3587
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3513..3532)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3673..3695
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3689..3880)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4689..5771)
                     /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     5733..5752
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5772..5849)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6055..6074
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6158..6177
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6158..6176
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6177..6201
                     /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             6216..6238
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg
       61 atggccatgg gccatcatca tcatcatcat catcatcatc acagcagcgg ccatatcgac
      121 gacgacgaca agcatatgca ggatgaaaaa gatccaaacc acattaaagt cggcgtgatt
      181 gttggtgccg aacagcaggt tgcagaagtc gcgcagaaag ttgcgaaaga caaatatggc
      241 ctggacgttg agctggtaac cttcaacgac tatgttctgc caaacgaagc attgagcaaa
      301 ggcgatatcg acgccaacgc cttccagcat aaaccgtacc ttgatcagca actgaaagat
      361 cgtggctaca aactggtcgc agtaggcaac acttttgttt atccgattgc tggttactcc
      421 aagaaaatca aatcactgga tgaactgcag gatggttcgc aggttgccgt gccaaacgac
      481 ccaactaacc ttggtcgttc actgctgctg ctgcaaaaag tgggcttgat caaactgaaa
      541 gatggcgttg gcctgctgcc gaccgttctt gatgttgttg agaaccccaa aaatctgaaa
      601 attgttgaac tggaagcacc gcaactgccg cgttctctgg acgacgcgca aatcgctctg
      661 gcagttatca ataccaccta tgccagccag attggcctga ctccggcgaa agacggtatc
      721 tttgttgaag ataaagagtc cccgtacgta gcgctgatcg tgacgcgtga agataacaaa
      781 gacgccgaga acgtgaagaa attcgtccag gcttatcagt ctgacgaagt ttacgaagca
      841 gcaaacaaag tgtttaacgg cggagctgtt aaaggctggt aactcgagca ccaccaccac
      901 caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
      961 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     1021 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa
     1081 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     1141 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     1201 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     1261 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     1321 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     1381 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     1441 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     1501 cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     1561 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     1621 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     1681 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     1741 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     1801 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     1861 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     1921 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     1981 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     2041 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     2101 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     2161 cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac
     2221 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     2281 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     2341 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     2401 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     2461 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     2521 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     2581 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     2641 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     2701 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     2761 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     2821 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     2881 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     2941 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     3001 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     3061 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     3121 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     3181 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     3241 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     3301 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     3361 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     3421 gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg
     3481 cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     3541 agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc
     3601 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     3661 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     3721 cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct
     3781 gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa
     3841 agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgccacc gtgtaagggg
     3901 gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg
     3961 ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat
     4021 ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag
     4081 atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg
     4141 tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc
     4201 atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta
     4261 tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg
     4321 acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct
     4381 cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc
     4441 cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga
     4501 aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca
     4561 taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg
     4621 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg
     4681 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     4741 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     4801 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     4861 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc
     4921 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca
     4981 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
     5041 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
     5101 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
     5161 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
     5221 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
     5281 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
     5341 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
     5401 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
     5461 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
     5521 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
     5581 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
     5641 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
     5701 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
     5761 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
     5821 cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc
     5881 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa
     5941 tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc
     6001 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat
     6061 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg
     6121 tccggcgtag aggatcgaga tctcgatccc gcgaaattaa tacgactcac tataggggaa
     6181 ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata
     6241 tacat
//
