LOCUS       sequence-228500-        6583 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N295A Mutation of MetN and M107A 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), M107A (EcI) MetNIQ in pBAD
FEATURES             Location/Qualifiers
     source          1..6583
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     107..126
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             224..253
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             269..283
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             2817..2834
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2883..2900)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3053..3139
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3231..3258
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3277..3368
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3369..3437,3438..4229)
                     /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(3587..3606)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4400..4988
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4889..4908
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5142..5159
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5174..5314
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5240..5259)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5400..5422
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5578..6456)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        join(6483..6583,1..184)
                     /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)"
ORIGIN
        1 ctgtaacaaa gcgggaccaa agccatgaca aaaacgcgta acaaaagtgt ctataatcac
       61 ggcagaaaag tccacattga ttatttgcac ggcgtcacac tttgctatgc catagcattt
      121 ttatccataa gattagcgga tcctacctga cgctttttat cgcaactctc tactgtttct
      181 ccatacccgt tttttgggct aacaggagga attaaccatg ggccatcatc atcatcatca
      241 tcatcatcat cacagcagcg gccatatcga cgacgacgac aagcatatga taaaactttc
      301 gaatatcacc aaagtgttcc accagggcac ccgcaccatc caggcgttga acaacgtcag
      361 cctgcatgtg ccagctggac aaatttatgg cgttatcggt gcctcaggcg cgggtaagag
      421 tacgcttata cgttgtgtaa acctgctgga gcgcccaacc gagggtagcg tgctggtcga
      481 tggccaggaa ctgaccacgc tgtcagaatc cgagttgacc aaagctcgcc gccagattgg
      541 tatgattttc cagcatttta acctgctctc ttcgcgtact gtttttggca acgtggctct
      601 gccgctggag ctggacaaca caccgaaaga cgaggtcaaa cgtcgcgtga cggaattgct
      661 gtcattggtt ggtcttggcg ataagcatga tagctacccg tcgaatcttt ccggtgggca
      721 gaaacaacgt gtggcaattg cccgtgcgtt agccagcaat cccaaagtat tgctgtgtga
      781 tgaagccacc agcgcgctgg acccggcaac gacacgttct attctcgaac tgctgaaaga
      841 catcaaccgc cgtctggggt tgacgattct gttgatcacc cacgaaatgg acgttgtgaa
      901 gcgcatttgt gattgcgtgg cggtcatcag caatggagaa ctgatcgagc aggacacggt
      961 aagtgaagtg ttctcgcatc cgaaaacgcc gctggcgcag aagtttattc agtcgaccct
     1021 gcatctggat atcccggaag attaccagga acgtctgcaa gcggagccat ttactgactg
     1081 cgtgccgatg ctgcgtctgg agtttaccgg tcaatcggtc gatgccccac tgctttctga
     1141 aaccgcgcgt cgtttcaacg tcaacaacgc gattattagc gcgcagatgg attacgccgg
     1201 tggcgttaag ttcggcatca tgctgactga aatgcacggc acacaacaag atacgcaagc
     1261 cgccattgcc tggctgcagg aacaccatgt aaaagtagag gtactgggtt atgtctgagc
     1321 cgatgatgtg gctgctggtt cgtggcgtat gggaaacgct ggcaatgacc ttcgtatccg
     1381 gtttttttgg ctttgtgatt ggtctgccgg ttggcgttct gctttatgtc acgcgtccgg
     1441 ggcaaattat tgctaacgcg aagctgtatc gtaccgtttc tgcgattgtg aacattttcc
     1501 gttccatccc gttcattatc ttgcttgtat ggatgattcc gtttacccgc gttattgtcg
     1561 gtacatcgat tggtttgcag gcagcgattg ttccgttaac cgttggtgca gcaccgttta
     1621 ttgcccgtgc ggtcgagaac gctctgctgg agatcccaac cgggttaatt gaagcttccc
     1681 gcgcaatggg tgccacgccg atgcagatcg tccgtaaggt gctgttaccg gaagcgctgc
     1741 cgggtctggt gaatgcggca actatcaccc tgattaccct ggtcggttat tccgcgatgg
     1801 gtggtgcagt cggtgccggt ggtttaggtc agattggcta tcagtatggc tacatcggct
     1861 ataacgcgac ggtgatgaat acggtactgg tattgctggt cattctggtt tatttaattc
     1921 agttcgcagg cgaccgcatc gtccgggctg tcactcgcaa gtaacgttca acacaacata
     1981 aataattgaa gaaggaataa ggtatggcgt tcaaattcaa aacctttgcg gcagtgggag
     2041 ccctgatcgg atcactggca ctggtaggct gcggtcagga tgaaaaagat ccaaaccaca
     2101 ttaaagtcgg cgtgattgtt ggtgccgaac agcaggttgc agaagtcgcg cagaaagttg
     2161 cgaaagacaa atatggcctg gacgttgagc tggtaacctt caacgactat gttctgccaa
     2221 acgaagcatt gagcaaaggc gatatcgacg ccaacgcctt ccagcataaa ccgtaccttg
     2281 atcagcaact gaaagatcgt ggctacaaac tggtcgcagt aggcaacact tttgtttatc
     2341 cgattgctgg ttactccaag aaaatcaaat cactggatga actgcaggat ggttcgcagg
     2401 ttgccgtgcc aaacgaccca actaaccttg gtcgttcact gctgctgctg caaaaagtgg
     2461 gcttgatcaa actgaaagat ggcgttggcc tgctgccgac cgttcttgat gttgttgaga
     2521 accccaaaaa tctgaaaatt gttgaactgg aagcaccgca actgccgcgt tctctggacg
     2581 acgcgcaaat cgctctggca gttatcaata ccacctatgc cagccagatt ggcctgactc
     2641 cggcgaaaga cggtatcttt gttgaagata aagagtcccc gtacgtaaac ctgatcgtga
     2701 cgcgtgaaga taacaaagac gccgagaacg tgaagaaatt cgtccaggct tatcagtctg
     2761 acgaagttta cgaagcagca aacaaagtgt ttaacggcgg agctgttaaa ggctggcacc
     2821 accaccacca ccactaagaa ttcgaagctt ggctgttttg gcggatgaga gaagattttc
     2881 agcctgatac agattaaatc agaacgcaga agcggtctga taaaacagaa tttgcctggc
     2941 ggcagtagcg cggtggtccc acctgacccc atgccgaact cagaagtgaa acgccgtagc
     3001 gccgatggta gtgtggggtc tccccatgcg agagtaggga actgccaggc atcaaataaa
     3061 acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc
     3121 tctcctgagt aggacaaatc cgccgggagc ggatttgaac gttgcgaagc aacggcccgg
     3181 agggtggcgg gcaggacgcc cgccataaac tgccaggcat caaattaagc agaaggccat
     3241 cctgacggat ggcctttttg cgtttctaca aactcttttg tttatttttc taaatacatt
     3301 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     3361 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     3421 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     3481 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     3541 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     3601 tattatcccg tgttgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     3661 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     3721 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     3781 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     3841 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     3901 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     3961 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     4021 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     4081 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     4141 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     4201 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     4261 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     4321 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     4381 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     4441 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     4501 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     4561 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     4621 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     4681 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     4741 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     4801 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     4861 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     4921 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     4981 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     5041 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     5101 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     5161 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     5221 gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     5281 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     5341 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     5401 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagcagat
     5461 caattcgcgc gcgaaggcga agcggcatgc ataatgtgcc tgtcaaatgg acgaagcagg
     5521 gattctgcaa accctatgct actccgtcaa gccgtcaatt gtctgattcg ttaccaatta
     5581 tgacaacttg acggctacat cattcacttt ttcttcacaa ccggcacgga actcgctcgg
     5641 gctggccccg gtgcattttt taaatacccg cgagaaatag agttgatcgt caaaaccaac
     5701 attgcgaccg acggtggcga taggcatccg ggtggtgctc aaaagcagct tcgcctggct
     5761 gatacgttgg tcctcgcgcc agcttaagac gctaatccct aactgctggc ggaaaagatg
     5821 tgacagacgc gacggcgaca agcaaacatg ctgtgcgacg ctggcgatat caaaattgct
     5881 gtctgccagg tgatcgctga tgtactgaca agcctcgcgt acccgattat ccatcggtgg
     5941 atggagcgac tcgttaatcg cttccatgcg ccgcagtaac aattgctcaa gcagatttat
     6001 cgccagcagc tccgaatagc gcccttcccc ttgcccggcg ttaatgattt gcccaaacag
     6061 gtcgctgaaa tgcggctggt gcgcttcatc cgggcgaaag aaccccgtat tggcaaatat
     6121 tgacggccag ttaagccatt catgccagta ggcgcgcgga cgaaagtaaa cccactggtg
     6181 ataccattcg cgagcctccg gatgacgacc gtagtgatga atctctcctg gcgggaacag
     6241 caaaatatca cccggtcggc aaacaaattc tcgtccctga tttttcacca ccccctgacc
     6301 gcgaatggtg agattgagaa tataaccttt cattcccagc ggtcggtcga taaaaaaatc
     6361 gagataaccg ttggcctcaa tcggcgttaa acccgccacc agatgggcat taaacgagta
     6421 tcccggcagc aggggatcat tttgcgcttc agccatactt ttcatactcc cgccattcag
     6481 agaagaaacc aattgtccat attgcatcag acattgccgt cactgcgtct tttactggct
     6541 cttctcgcta accaaaccgg taaccccgct tattaaaagc att
//
