LOCUS       sequence-223951-        6583 bp    DNA     circular SYN 15-APR-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 Apr 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"
     promoter        1..285
                     /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     208..227
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             325..354
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             370..384
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             2918..2935
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2984..3001)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3154..3240
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3332..3359
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3378..3469
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3470..3538,3539..4330)
                     /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(3688..3707)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4501..5089
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4990..5009
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5243..5260
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5275..5415
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5341..5360)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5501..5523
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5679..6557)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct
       61 tctcgctaac caaaccggta accccgctta ttaaaagcat tctgtaacaa agcgggacca
      121 aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg
      181 attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg
      241 atcctacctg acgcttttta tcgcaactct ctactgtttc tccatacccg ttttttgggc
      301 taacaggagg aattaaccat gggccatcat catcatcatc atcatcatca tcacagcagc
      361 ggccatatcg acgacgacga caagcatatg ataaaacttt cgaatatcac caaagtgttc
      421 caccagggca cccgcaccat ccaggcgttg aacaacgtca gcctgcatgt gccagctgga
      481 caaatttatg gcgttatcgg tgcctcaggc gcgggtaaga gtacgcttat acgttgtgta
      541 aacctgctgg agcgcccaac cgagggtagc gtgctggtcg atggccagga actgaccacg
      601 ctgtcagaat ccgagttgac caaagctcgc cgccagattg gtatgatttt ccagcatttt
      661 aacctgctct cttcgcgtac tgtttttggc aacgtggctc tgccgctgga gctggacaac
      721 acaccgaaag acgaggtcaa acgtcgcgtg acggaattgc tgtcattggt tggtcttggc
      781 gataagcatg atagctaccc gtcgaatctt tccggtgggc agaaacaacg tgtggcaatt
      841 gcccgtgcgt tagccagcaa tcccaaagta ttgctgtgtg atgaagccac cagcgcgctg
      901 gacccggcaa cgacacgttc tattctcgaa ctgctgaaag acatcaaccg ccgtctgggg
      961 ttgacgattc tgttgatcac ccacgaaatg gacgttgtga agcgcatttg tgattgcgtg
     1021 gcggtcatca gcaatggaga actgatcgag caggacacgg taagtgaagt gttctcgcat
     1081 ccgaaaacgc cgctggcgca gaagtttatt cagtcgaccc tgcatctgga tatcccggaa
     1141 gattaccagg aacgtctgca agcggagcca tttactgact gcgtgccgat gctgcgtctg
     1201 gagtttaccg gtcaatcggt cgatgcccca ctgctttctg aaaccgcgcg tcgtttcaac
     1261 gtcaacaacg ccattattag cgcgcagatg gattacgccg gtggcgttaa gttcggcatc
     1321 atgctgactg aaatgcacgg cacacaacaa gatacgcaag ccgccattgc ctggctgcag
     1381 gaacaccatg taaaagtaga ggtactgggt tatgtctgag ccgatgatgt ggctgctggt
     1441 tcgtggcgta tgggaaacgc tggcaatgac cttcgtatcc ggtttttttg gctttgtgat
     1501 tggtctgccg gttggcgttc tgctttatgt cacgcgtccg gggcaaatta ttgctaacgc
     1561 gaagctgtat cgtaccgttt ctgcgattgt gaacattttc cgttccatcc cgttcattat
     1621 cttgcttgta tggatgattc cgtttacccg cgttattgtc ggtacatcga ttggtttgca
     1681 ggcagcgatt gttccgttaa ccgttggtgc agcaccgttt attgcccgta tggtcgagaa
     1741 cgctctgctg gagatcccaa ccgggttaat tgaagcttcc cgcgcaatgg gtgccacgcc
     1801 gatgcagatc gtccgtaagg tgctgttacc ggaagcgctg ccgggtctgg tgaatgcggc
     1861 aactatcacc ctgattaccc tggtcggtta ttccgcgatg ggtggtgcag tcggtgccgg
     1921 tggtttaggt cagattggct atcagtatgg ctacatcggc tataacgcga cggtgatgaa
     1981 tacggtactg gtattgctgg tcattctggt ttatttaatt cagttcgcag gcgaccgcat
     2041 cgtccgggct gtcactcgca agtaacgttc aacacaacat aaataattga agaaggaata
     2101 aggtatggcg ttcaaattca aaacctttgc ggcagtggga gccctgatcg gatcactggc
     2161 actggtaggc tgcggtcagg atgaaaaaga tccaaaccac attaaagtcg gcgtgattgt
     2221 tggtgccgaa cagcaggttg cagaagtcgc gcagaaagtt gcgaaagaca aatatggcct
     2281 ggacgttgag ctggtaacct tcaacgacta tgttctgcca aacgaagcat tgagcaaagg
     2341 cgatatcgac gccaacgcct tccagcataa accgtacctt gatcagcaac tgaaagatcg
     2401 tggctacaaa ctggtcgcag taggcaacac ttttgtttat ccgattgctg gttactccaa
     2461 gaaaatcaaa tcactggatg aactgcagga tggttcgcag gttgccgtgc caaacgaccc
     2521 aactaacctt ggtcgttcac tgctgctgct gcaaaaagtg ggcttgatca aactgaaaga
     2581 tggcgttggc ctgctgccga ccgttcttga tgttgttgag aaccccaaaa atctgaaaat
     2641 tgttgaactg gaagcaccgc aactgccgcg ttctctggac gacgcgcaaa tcgctctggc
     2701 agttatcaat accacctatg ccagccagat tggcctgact ccggcgaaag acggtatctt
     2761 tgttgaagat aaagagtccc cgtacgtagc cctgatcgtg acgcgtgaag ataacaaaga
     2821 cgccgagaac gtgaagaaat tcgtccaggc ttatcagtct gacgaagttt acgaagcagc
     2881 aaacaaagtg tttaacggcg gagctgttaa aggctggcac caccaccacc accactaaga
     2941 attcgaagct tggctgtttt ggcggatgag agaagatttt cagcctgata cagattaaat
     3001 cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc
     3061 cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt
     3121 ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa
     3181 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat
     3241 ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg ggcaggacgc
     3301 ccgccataaa ctgccaggca tcaaattaag cagaaggcca tcctgacgga tggccttttt
     3361 gcgtttctac aaactctttt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     3421 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     3481 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     3541 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     3601 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     3661 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc
     3721 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     3781 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     3841 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     3901 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     3961 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     4021 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     4081 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     4141 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     4201 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     4261 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     4321 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     4381 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     4441 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     4501 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     4561 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     4621 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     4681 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     4741 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     4801 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     4861 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     4921 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     4981 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     5041 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     5101 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     5161 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     5221 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     5281 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag
     5341 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
     5401 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     5461 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     5521 aggttttcac cgtcatcacc gaaacgcgcg aggcagcaga tcaattcgcg cgcgaaggcg
     5581 aagcggcatg cataatgtgc ctgtcaaatg gacgaagcag ggattctgca aaccctatgc
     5641 tactccgtca agccgtcaat tgtctgattc gttaccaatt atgacaactt gacggctaca
     5701 tcattcactt tttcttcaca accggcacgg aactcgctcg ggctggcccc ggtgcatttt
     5761 ttaaataccc gcgagaaata gagttgatcg tcaaaaccaa cattgcgacc gacggtggcg
     5821 ataggcatcc gggtggtgct caaaagcagc ttcgcctggc tgatacgttg gtcctcgcgc
     5881 cagcttaaga cgctaatccc taactgctgg cggaaaagat gtgacagacg cgacggcgac
     5941 aagcaaacat gctgtgcgac gctggcgata tcaaaattgc tgtctgccag gtgatcgctg
     6001 atgtactgac aagcctcgcg tacccgatta tccatcggtg gatggagcga ctcgttaatc
     6061 gcttccatgc gccgcagtaa caattgctca agcagattta tcgccagcag ctccgaatag
     6121 cgcccttccc cttgcccggc gttaatgatt tgcccaaaca ggtcgctgaa atgcggctgg
     6181 tgcgcttcat ccgggcgaaa gaaccccgta ttggcaaata ttgacggcca gttaagccat
     6241 tcatgccagt aggcgcgcgg acgaaagtaa acccactggt gataccattc gcgagcctcc
     6301 ggatgacgac cgtagtgatg aatctctcct ggcgggaaca gcaaaatatc acccggtcgg
     6361 caaacaaatt ctcgtccctg atttttcacc accccctgac cgcgaatggt gagattgaga
     6421 atataacctt tcattcccag cggtcggtcg ataaaaaaat cgagataacc gttggcctca
     6481 atcggcgtta aacccgccac cagatgggca ttaaacgagt atcccggcag caggggatca
     6541 ttttgcgctt cagccatact tttcatactc ccgccattca gag
//
