LOCUS       sequence-228498-        6583 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N295A Mutation of MetN and F103A in MetI in Methionine ABC 
            Transporter and Wild Type Methionine Binding Protein 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), F103A (EcI) MetNIQ in pBAD
FEATURES             Location/Qualifiers
     source          1..6583
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2344..2361
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2410..2427)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2580..2666
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2758..2785
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2804..2895
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2896..2964,2965..3756)
                     /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(3114..3133)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3927..4515
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4416..4435
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4669..4686
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4701..4841
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4767..4786)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4927..4949
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5105..5983)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        6010..6294
                     /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     6217..6236
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             6334..6363
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             6379..6393
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
ORIGIN
        1 tggtcgatgg ccaggaactg accacgctgt cagaatccga gttgaccaaa gctcgccgcc
       61 agattggtat gattttccag cattttaacc tgctctcttc gcgtactgtt tttggcaacg
      121 tggctctgcc gctggagctg gacaacacac cgaaagacga ggtcaaacgt cgcgtgacgg
      181 aattgctgtc attggttggt cttggcgata agcatgatag ctacccgtcg aatctttccg
      241 gtgggcagaa acaacgtgtg gcaattgccc gtgcgttagc cagcaatccc aaagtattgc
      301 tgtgtgatga agccaccagc gcgctggacc cggcaacgac acgttctatt ctcgaactgc
      361 tgaaagacat caaccgccgt ctggggttga cgattctgtt gatcacccac gaaatggacg
      421 ttgtgaagcg catttgtgat tgcgtggcgg tcatcagcaa tggagaactg atcgagcagg
      481 acacggtaag tgaagtgttc tcgcatccga aaacgccgct ggcgcagaag tttattcagt
      541 cgaccctgca tctggatatc ccggaagatt accaggaacg tctgcaagcg gagccattta
      601 ctgactgcgt gccgatgctg cgtctggagt ttaccggtca atcggtcgat gccccactgc
      661 tttctgaaac cgcgcgtcgt ttcaacgtca acaacgcgat tattagcgcg cagatggatt
      721 acgccggtgg cgttaagttc ggcatcatgc tgactgaaat gcacggcaca caacaagata
      781 cgcaagccgc cattgcctgg ctgcaggaac accatgtaaa agtagaggta ctgggttatg
      841 tctgagccga tgatgtggct gctggttcgt ggcgtatggg aaacgctggc aatgaccttc
      901 gtatccggtt tttttggctt tgtgattggt ctgccggttg gcgttctgct ttatgtcacg
      961 cgtccggggc aaattattgc taacgcgaag ctgtatcgta ccgtttctgc gattgtgaac
     1021 attttccgtt ccatcccgtt cattatcttg cttgtatgga tgattccgtt tacccgcgtt
     1081 attgtcggta catcgattgg tttgcaggca gcgattgttc cgttaaccgt tggtgcagca
     1141 ccggcgattg cccgtatggt cgagaacgct ctgctggaga tcccaaccgg gttaattgaa
     1201 gcttcccgcg caatgggtgc cacgccgatg cagatcgtcc gtaaggtgct gttaccggaa
     1261 gcgctgccgg gtctggtgaa tgcggcaact atcaccctga ttaccctggt cggttattcc
     1321 gcgatgggtg gtgcagtcgg tgccggtggt ttaggtcaga ttggctatca gtatggctac
     1381 atcggctata acgcgacggt gatgaatacg gtactggtat tgctggtcat tctggtttat
     1441 ttaattcagt tcgcaggcga ccgcatcgtc cgggctgtca ctcgcaagta acgttcaaca
     1501 caacataaat aattgaagaa ggaataaggt atggcgttca aattcaaaac ctttgcggca
     1561 gtgggagccc tgatcggatc actggcactg gtaggctgcg gtcaggatga aaaagatcca
     1621 aaccacatta aagtcggcgt gattgttggt gccgaacagc aggttgcaga agtcgcgcag
     1681 aaagttgcga aagacaaata tggcctggac gttgagctgg taaccttcaa cgactatgtt
     1741 ctgccaaacg aagcattgag caaaggcgat atcgacgcca acgccttcca gcataaaccg
     1801 taccttgatc agcaactgaa agatcgtggc tacaaactgg tcgcagtagg caacactttt
     1861 gtttatccga ttgctggtta ctccaagaaa atcaaatcac tggatgaact gcaggatggt
     1921 tcgcaggttg ccgtgccaaa cgacccaact aaccttggtc gttcactgct gctgctgcaa
     1981 aaagtgggct tgatcaaact gaaagatggc gttggcctgc tgccgaccgt tcttgatgtt
     2041 gttgagaacc ccaaaaatct gaaaattgtt gaactggaag caccgcaact gccgcgttct
     2101 ctggacgacg cgcaaatcgc tctggcagtt atcaatacca cctatgccag ccagattggc
     2161 ctgactccgg cgaaagacgg tatctttgtt gaagataaag agtccccgta cgtaaacctg
     2221 atcgtgacgc gtgaagataa caaagacgcc gagaacgtga agaaattcgt ccaggcttat
     2281 cagtctgacg aagtttacga agcagcaaac aaagtgttta acggcggagc tgttaaaggc
     2341 tggcaccacc accaccacca ctaagaattc gaagcttggc tgttttggcg gatgagagaa
     2401 gattttcagc ctgatacaga ttaaatcaga acgcagaagc ggtctgataa aacagaattt
     2461 gcctggcggc agtagcgcgg tggtcccacc tgaccccatg ccgaactcag aagtgaaacg
     2521 ccgtagcgcc gatggtagtg tggggtctcc ccatgcgaga gtagggaact gccaggcatc
     2581 aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg
     2641 tgaacgctct cctgagtagg acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac
     2701 ggcccggagg gtggcgggca ggacgcccgc cataaactgc caggcatcaa attaagcaga
     2761 aggccatcct gacggatggc ctttttgcgt ttctacaaac tcttttgttt atttttctaa
     2821 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     2881 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     2941 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     3001 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     3061 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     3121 ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat
     3181 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     3241 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     3301 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     3361 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     3421 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     3481 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     3541 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     3601 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     3661 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     3721 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     3781 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     3841 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     3901 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     3961 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     4021 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     4081 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     4141 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     4201 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     4261 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     4321 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     4381 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     4441 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     4501 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     4561 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     4621 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     4681 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     4741 tcacaccgca tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     4801 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
     4861 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     4921 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
     4981 agcagatcaa ttcgcgcgcg aaggcgaagc ggcatgcata atgtgcctgt caaatggacg
     5041 aagcagggat tctgcaaacc ctatgctact ccgtcaagcc gtcaattgtc tgattcgtta
     5101 ccaattatga caacttgacg gctacatcat tcactttttc ttcacaaccg gcacggaact
     5161 cgctcgggct ggccccggtg cattttttaa atacccgcga gaaatagagt tgatcgtcaa
     5221 aaccaacatt gcgaccgacg gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg
     5281 cctggctgat acgttggtcc tcgcgccagc ttaagacgct aatccctaac tgctggcgga
     5341 aaagatgtga cagacgcgac ggcgacaagc aaacatgctg tgcgacgctg gcgatatcaa
     5401 aattgctgtc tgccaggtga tcgctgatgt actgacaagc ctcgcgtacc cgattatcca
     5461 tcggtggatg gagcgactcg ttaatcgctt ccatgcgccg cagtaacaat tgctcaagca
     5521 gatttatcgc cagcagctcc gaatagcgcc cttccccttg cccggcgtta atgatttgcc
     5581 caaacaggtc gctgaaatgc ggctggtgcg cttcatccgg gcgaaagaac cccgtattgg
     5641 caaatattga cggccagtta agccattcat gccagtaggc gcgcggacga aagtaaaccc
     5701 actggtgata ccattcgcga gcctccggat gacgaccgta gtgatgaatc tctcctggcg
     5761 ggaacagcaa aatatcaccc ggtcggcaaa caaattctcg tccctgattt ttcaccaccc
     5821 cctgaccgcg aatggtgaga ttgagaatat aacctttcat tcccagcggt cggtcgataa
     5881 aaaaatcgag ataaccgttg gcctcaatcg gcgttaaacc cgccaccaga tgggcattaa
     5941 acgagtatcc cggcagcagg ggatcatttt gcgcttcagc catacttttc atactcccgc
     6001 cattcagaga agaaaccaat tgtccatatt gcatcagaca ttgccgtcac tgcgtctttt
     6061 actggctctt ctcgctaacc aaaccggtaa ccccgcttat taaaagcatt ctgtaacaaa
     6121 gcgggaccaa agccatgaca aaaacgcgta acaaaagtgt ctataatcac ggcagaaaag
     6181 tccacattga ttatttgcac ggcgtcacac tttgctatgc catagcattt ttatccataa
     6241 gattagcgga tcctacctga cgctttttat cgcaactctc tactgtttct ccatacccgt
     6301 tttttgggct aacaggagga attaaccatg ggccatcatc atcatcatca tcatcatcat
     6361 cacagcagcg gccatatcga cgacgacgac aagcatatga taaaactttc gaatatcacc
     6421 aaagtgttcc accagggcac ccgcaccatc caggcgttga acaacgtcag cctgcatgtg
     6481 ccagctggac aaatttatgg cgttatcggt gcctcaggcg cgggtaagag tacgcttata
     6541 cgttgtgtaa acctgctgga gcgcccaacc gagggtagcg tgc
//
