LOCUS       sequence-228549-        6583 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N295A Mutation of MetN in Methionine ABC Transporter and N229A 
            Mutation in Methionine Binding Protein (MetQ) for Transport Activity
            Assay.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6583)
  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 6583)
  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: N295A (EcN), N229A (EcQ) MetNIQ in pBAD
FEATURES             Location/Qualifiers
     source          1..6583
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             40..69
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             85..99
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             2633..2650
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2699..2716)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2869..2955
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3047..3074
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3093..3184
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3185..3253,3254..4045)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3403..3422)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4216..4804
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4705..4724
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4958..4975
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4990..5130
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5056..5075)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5216..5238
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5394..6272)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        6299..6583
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     6506..6525
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
ORIGIN
        1 acccgttttt tgggctaaca ggaggaatta accatgggcc atcatcatca tcatcatcat
       61 catcatcaca gcagcggcca tatcgacgac gacgacaagc atatgataaa actttcgaat
      121 atcaccaaag tgttccacca gggcacccgc accatccagg cgttgaacaa cgtcagcctg
      181 catgtgccag ctggacaaat ttatggcgtt atcggtgcct caggcgcggg taagagtacg
      241 cttatacgtt gtgtaaacct gctggagcgc ccaaccgagg gtagcgtgct ggtcgatggc
      301 caggaactga ccacgctgtc agaatccgag ttgaccaaag ctcgccgcca gattggtatg
      361 attttccagc attttaacct gctctcttcg cgtactgttt ttggcaacgt ggctctgccg
      421 ctggagctgg acaacacacc gaaagacgag gtcaaacgtc gcgtgacgga attgctgtca
      481 ttggttggtc ttggcgataa gcatgatagc tacccgtcga atctttccgg tgggcagaaa
      541 caacgtgtgg caattgcccg tgcgttagcc agcaatccca aagtattgct gtgtgatgaa
      601 gccaccagcg cgctggaccc ggcaacgaca cgttctattc tcgaactgct gaaagacatc
      661 aaccgccgtc tggggttgac gattctgttg atcacccacg aaatggacgt tgtgaagcgc
      721 atttgtgatt gcgtggcggt catcagcaat ggagaactga tcgagcagga cacggtaagt
      781 gaagtgttct cgcatccgaa aacgccgctg gcgcagaagt ttattcagtc gaccctgcat
      841 ctggatatcc cggaagatta ccaggaacgt ctgcaagcgg agccatttac tgactgcgtg
      901 ccgatgctgc gtctggagtt taccggtcaa tcggtcgatg ccccactgct ttctgaaacc
      961 gcgcgtcgtt tcaacgtcaa caacgcgatt attagcgcgc agatggatta cgccggtggc
     1021 gttaagttcg gcatcatgct gactgaaatg cacggcacac aacaagatac gcaagccgcc
     1081 attgcctggc tgcaggaaca ccatgtaaaa gtagaggtac tgggttatgt ctgagccgat
     1141 gatgtggctg ctggttcgtg gcgtatggga aacgctggca atgaccttcg tatccggttt
     1201 ttttggcttt gtgattggtc tgccggttgg cgttctgctt tatgtcacgc gtccggggca
     1261 aattattgct aacgcgaagc tgtatcgtac cgtttctgcg attgtgaaca ttttccgttc
     1321 catcccgttc attatcttgc ttgtatggat gattccgttt acccgcgtta ttgtcggtac
     1381 atcgattggt ttgcaggcag cgattgttcc gttaaccgtt ggtgcagcac cgtttattgc
     1441 ccgtatggtc gagaacgctc tgctggagat cccaaccggg ttaattgaag cttcccgcgc
     1501 aatgggtgcc acgccgatgc agatcgtccg taaggtgctg ttaccggaag cgctgccggg
     1561 tctggtgaat gcggcaacta tcaccctgat taccctggtc ggttattccg cgatgggtgg
     1621 tgcagtcggt gccggtggtt taggtcagat tggctatcag tatggctaca tcggctataa
     1681 cgcgacggtg atgaatacgg tactggtatt gctggtcatt ctggtttatt taattcagtt
     1741 cgcaggcgac cgcatcgtcc gggctgtcac tcgcaagtaa cgttcaacac aacataaata
     1801 attgaagaag gaataaggta tggcgttcaa attcaaaacc tttgcggcag tgggagccct
     1861 gatcggatca ctggcactgg taggctgcgg tcaggatgaa aaagatccaa accacattaa
     1921 agtcggcgtg attgttggtg ccgaacagca ggttgcagaa gtcgcgcaga aagttgcgaa
     1981 agacaaatat ggcctggacg ttgagctggt aaccttcaac gactatgttc tgccaaacga
     2041 agcattgagc aaaggcgata tcgacgccaa cgccttccag cataaaccgt accttgatca
     2101 gcaactgaaa gatcgtggct acaaactggt cgcagtaggc aacacttttg tttatccgat
     2161 tgctggttac tccaagaaaa tcaaatcact ggatgaactg caggatggtt cgcaggttgc
     2221 cgtgccaaac gacccaacta accttggtcg ttcactgctg ctgctgcaaa aagtgggctt
     2281 gatcaaactg aaagatggcg ttggcctgct gccgaccgtt cttgatgttg ttgagaaccc
     2341 caaaaatctg aaaattgttg aactggaagc accgcaactg ccgcgttctc tggacgacgc
     2401 gcaaatcgct ctggcagtta tcaataccac ctatgccagc cagattggcc tgactccggc
     2461 gaaagacggt atctttgttg aagataaaga gtccccgtac gtagccctga tcgtgacgcg
     2521 tgaagataac aaagacgccg agaacgtgaa gaaattcgtc caggcttatc agtctgacga
     2581 agtttacgaa gcagcaaaca aagtgtttaa cggcggagct gttaaaggct ggcaccacca
     2641 ccaccaccac taagaattcg aagcttggct gttttggcgg atgagagaag attttcagcc
     2701 tgatacagat taaatcagaa cgcagaagcg gtctgataaa acagaatttg cctggcggca
     2761 gtagcgcggt ggtcccacct gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg
     2821 atggtagtgt ggggtctccc catgcgagag tagggaactg ccaggcatca aataaaacga
     2881 aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc
     2941 ctgagtagga caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg
     3001 tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg
     3061 acggatggcc tttttgcgtt tctacaaact cttttgttta tttttctaaa tacattcaaa
     3121 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     3181 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     3241 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     3301 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     3361 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     3421 atcccgtgtt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     3481 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     3541 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     3601 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     3661 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     3721 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     3781 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     3841 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     3901 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     3961 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     4021 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     4081 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     4141 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     4201 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     4261 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     4321 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     4381 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     4441 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     4501 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     4561 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     4621 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     4681 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     4741 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     4801 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     4861 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     4921 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     4981 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     5041 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     5101 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     5161 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     5221 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gcagatcaat
     5281 tcgcgcgcga aggcgaagcg gcatgcataa tgtgcctgtc aaatggacga agcagggatt
     5341 ctgcaaaccc tatgctactc cgtcaagccg tcaattgtct gattcgttac caattatgac
     5401 aacttgacgg ctacatcatt cactttttct tcacaaccgg cacggaactc gctcgggctg
     5461 gccccggtgc attttttaaa tacccgcgag aaatagagtt gatcgtcaaa accaacattg
     5521 cgaccgacgg tggcgatagg catccgggtg gtgctcaaaa gcagcttcgc ctggctgata
     5581 cgttggtcct cgcgccagct taagacgcta atccctaact gctggcggaa aagatgtgac
     5641 agacgcgacg gcgacaagca aacatgctgt gcgacgctgg cgatatcaaa attgctgtct
     5701 gccaggtgat cgctgatgta ctgacaagcc tcgcgtaccc gattatccat cggtggatgg
     5761 agcgactcgt taatcgcttc catgcgccgc agtaacaatt gctcaagcag atttatcgcc
     5821 agcagctccg aatagcgccc ttccccttgc ccggcgttaa tgatttgccc aaacaggtcg
     5881 ctgaaatgcg gctggtgcgc ttcatccggg cgaaagaacc ccgtattggc aaatattgac
     5941 ggccagttaa gccattcatg ccagtaggcg cgcggacgaa agtaaaccca ctggtgatac
     6001 cattcgcgag cctccggatg acgaccgtag tgatgaatct ctcctggcgg gaacagcaaa
     6061 atatcacccg gtcggcaaac aaattctcgt ccctgatttt tcaccacccc ctgaccgcga
     6121 atggtgagat tgagaatata acctttcatt cccagcggtc ggtcgataaa aaaatcgaga
     6181 taaccgttgg cctcaatcgg cgttaaaccc gccaccagat gggcattaaa cgagtatccc
     6241 ggcagcaggg gatcattttg cgcttcagcc atacttttca tactcccgcc attcagagaa
     6301 gaaaccaatt gtccatattg catcagacat tgccgtcact gcgtctttta ctggctcttc
     6361 tcgctaacca aaccggtaac cccgcttatt aaaagcattc tgtaacaaag cgggaccaaa
     6421 gccatgacaa aaacgcgtaa caaaagtgtc tataatcacg gcagaaaagt ccacattgat
     6481 tatttgcacg gcgtcacact ttgctatgcc atagcatttt tatccataag attagcggat
     6541 cctacctgac gctttttatc gcaactctct actgtttctc cat
//
