LOCUS       sequence-223944-        7426 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N295A and E166Q Mutation of MetN in Methionine ABC Transporter with 
            6x His Tag. Used to solve crystal structure.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7426)
  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 7426)
  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), E166Q (EcN) MetNI LmH
FEATURES             Location/Qualifiers
     source          1..7426
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     296..315
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     549..566
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    581..723
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(649..668)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     809..831
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(825..1016)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2328..3410)
                     /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     3372..3391
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3411..3488)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        3604..3622
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3623..3647
                     /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             3662..3684
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4349..4366
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      4433..4480
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        4522..4540
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4541..4565
                     /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             4580..4602
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4615..4632
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      5734..5781
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(5956..5961,5962..5978,5979..5984))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(6011..6029)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6097..6201
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6202..6270,6271..7062)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6420..6439)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7233..7426,1..395)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
       61 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
      121 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
      181 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
      241 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
      301 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
      361 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
      421 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
      481 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
      541 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
      601 ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta caatctgctc
      661 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct
      721 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
      781 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
      841 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat
      901 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat
      961 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat
     1021 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg
     1081 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt
     1141 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag
     1201 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag
     1261 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg
     1321 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     1381 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
     1441 agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggccagga cccaacgctg
     1501 cccgagatgc gccgcgtgcg gctgctggag atggcggacg cgatggatat gttctgccaa
     1561 gggttggttt gcgcattcac agttctccgc aagaattgat tggctccaat tcttggagtg
     1621 gtgaatccgt tagcgaggtg ccgccggctt ccattcaggt cgaggtggcc cggctccatg
     1681 caccgcgacg caacgcgggg aggcagacaa ggtatagggc ggcgcctaca atccatgcca
     1741 acccgttcca tgtgctcgcc gaggcggcat aaatcgccgt gacgatcagc ggtccagtga
     1801 tcgaagttag gctggtaaga gccgcgagcg atccttgaag ctgtccctga tggtcgtcat
     1861 ctacctgcct ggacagcatg gcctgcaacg cgggcatccc gatgccgccg gaagcgagaa
     1921 gaatcataat ggggaaggcc atccagcctc gcgtcgcgaa cgccagcaag acgtagccca
     1981 gcgcgtcggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg
     2041 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc
     2101 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg
     2161 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca
     2221 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag
     2281 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt
     2341 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     2401 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc
     2461 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc
     2521 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct
     2581 tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta
     2641 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg
     2701 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct
     2761 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga
     2821 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc
     2881 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg
     2941 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct
     3001 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt
     3061 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc
     3121 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc
     3181 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc
     3241 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact
     3301 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga
     3361 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc
     3421 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg
     3481 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag
     3541 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgcc gatcccgcga
     3601 aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa
     3661 ttttgtttaa ctttaagaag gagatataca tatgtctgag ccgatgatgt ggctgctggt
     3721 tcgtggcgta tgggaaacgc tggcaatgac cttcgtatcc ggtttttttg gctttgtgat
     3781 tggtctgccg gttggcgttc tgctttatgt cacgcgtccg gggcaaatta ttgctaacgc
     3841 gaagctgtat cgtaccgttt ctgcgattgt gaacattttc cgttccatcc cgttcattat
     3901 cttgcttgta tggatgattc cgtttacccg cgttattgtc ggtacatcga ttggtttgca
     3961 ggcagcgatt gttccgttaa ccgttggtgc agcaccgttt attgcccgta tggtcgagaa
     4021 cgctctgctg gagatcccaa ccgggttaat tgaagcttcc cgcgcaatgg gtgccacgcc
     4081 gatgcagatc gtccgtaagg tgctgttacc ggaagcgctg ccgggtctgg tgaatgcggc
     4141 aactatcacc ctgattaccc tggtcggtta ttccgcgatg ggtggtgcag tcggtgccgg
     4201 tggtttaggt cagattggct atcagtatgg ctacatcggc tataacgcga cggtgatgaa
     4261 tacggtactg gtattgctgg tcattctggt ttatttaatt cagttcgcag gcgaccgcat
     4321 cgtccgggct gtcactcgca agtgagagca ccaccaccac caccactgag atccggctgc
     4381 taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata
     4441 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc
     4501 cagatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca
     4561 attcccctct agaaataatt ttgtttaact ttaagaagga gatataccat gggccatcat
     4621 catcatcatc atatgataaa actttcgaat atcaccaaag tgttccacca gggcacccgc
     4681 accatccagg cgttgaacaa cgtcagcctg catgtgccag ctggacaaat ttatggcgtt
     4741 atcggtgcct caggcgcggg taagagtacg cttatacgtt gtgtaaacct gctggagcgc
     4801 ccaaccgagg gtagcgtgct ggtcgatggc caggaactga ccacgctgtc agaatccgag
     4861 ttgaccaaag ctcgccgcca gattggtatg attttccagc attttaacct gctctcttcg
     4921 cgtactgttt ttggcaacgt ggctctgccg ctggagctgg acaacacacc gaaagacgag
     4981 gtcaaacgtc gcgtgacgga attgctgtca ttggttggtc ttggcgataa gcatgatagc
     5041 tacccgtcga atctttccgg tgggcagaaa caacgtgtgg caattgcccg tgcgttagcc
     5101 agcaatccca aagtattgct gtgtgatcaa gccaccagcg cgctggaccc ggcaacgaca
     5161 cgttctattc tcgaactgct gaaagacatc aaccgccgtc tggggttgac gattctgttg
     5221 atcacccacg aaatggacgt tgtgaagcgc atttgtgatt gcgtggcggt catcagcaat
     5281 ggagaactga tcgagcagga cacggtaagt gaagtgttct cgcatccgaa aacgccgctg
     5341 gcgcagaagt ttattcagtc gaccctgcat ctggatatcc cggaagatta ccaggaacgt
     5401 ctgcaagcgg agccatttac tgactgcgtg ccgatgctgc gtctggagtt taccggtcaa
     5461 tcggtcgatg ccccactgct ttctgaaacc gcgcgtcgtt tcaacgtcaa caacgcgatt
     5521 attagcgcgc agatggatta cgccggtggc gttaagttcg gcatcatgct gactgaaatg
     5581 cacggcacac aacaagatac gcaagccgcc attgcctggc tgcaggaaca ccatgtaaaa
     5641 gtagaggtac tgggttatgt ctgactcgag gatccggctg ctaacaaagc ccgaaaggaa
     5701 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
     5761 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggatatcc cgcaagaggc
     5821 ccggcagtac cggcataacc aagcctatgc ctacagcatc cagggtgacg gtgccgagga
     5881 tgacgatgag cgcattgtta gatttcatac acggtgcctg actgcgttag caatttaact
     5941 gtgataaact accgcattaa agcttatcga tgataagctg tcaaacatga gaattcttga
     6001 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     6061 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     6121 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     6181 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     6241 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     6301 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     6361 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     6421 ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata
     6481 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     6541 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     6601 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     6661 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     6721 gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact
     6781 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     6841 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     6901 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     6961 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     7021 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     7081 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     7141 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     7201 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     7261 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     7321 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     7381 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtag
//
