LOCUS       sequence-228542-        7423 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 7423)
  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 7423)
  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..7423
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1..23
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             688..705
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      772..819
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        861..879
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    880..904
                     /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             919..941
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             954..971
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2073..2120
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2295..2300,2301..2317,2318..2323))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2350..2368)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2436..2540
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2541..2609,2610..3401)
                     /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(2759..2778)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3572..4160
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4061..4080
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4314..4331
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4346..4485
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4411..4430)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4571..4593
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4587..4778)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6090..7172)
                     /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     7134..7153
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7173..7250)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        7366..7384
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7385..7409
                     /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)."
ORIGIN
        1 tttgtttaac tttaagaagg agatatacat atgtctgagc cgatgatgtg gctgctggtt
       61 cgtggcgtat gggaaacgct ggcaatgacc ttcgtatccg gtttttttgg ctttgtgatt
      121 ggtctgccgg ttggcgttct gctttatgtc acgcgtccgg ggcaaattat tgctaacgcg
      181 aagctgtatc gtaccgtttc tgcgattgtg aacattttcc gttccatccc gttcattatc
      241 ttgcttgtat ggatgattcc gtttacccgc gttattgtcg gtacatcgat tggtttgcag
      301 gcagcgattg ttccgttaac cgttggtgca gcaccgttta ttgcccgtat ggtcgagaac
      361 gctctgctgg agatcccaac cgggttaatt gaagcttccc gcgcaatggg tgccacgccg
      421 atgcagatcg tccgtaaggt gctgttaccg gaagcgctgc cgggtctggt gaatgcggca
      481 actatcaccc tgattaccct ggtcggttat tccgcgatgg gtggtgcagt cggtgccggt
      541 ggtttaggtc agattggcta tcagtatggc tacatcggct ataacgcgac ggtgatgaat
      601 acggtactgg tattgctggt cattctggtt tatttaattc agttcgcagg cgaccgcatc
      661 gtccgggctg tcactcgcaa gtgagagcac caccaccacc accactgaga tccggctgct
      721 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
      781 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
      841 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
      901 ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg ggccatcatc
      961 atcatcatca tatgataaaa ctttcgaata tcaccaaagt gttccaccag ggcacccgca
     1021 ccatccaggc gttgaacaac gtcagcctgc atgtgccagc tggacaaatt tatggcgtta
     1081 tcggtgcctc aggcgcgggt aagagtacgc ttatacgttg tgtaaacctg ctggagcgcc
     1141 caaccgaggg tagcgtgctg gtcgatggcc aggaactgac cacgctgtca gaatccgagt
     1201 tgaccaaagc tcgccgccag attggtatga ttttccagca ttttaacctg ctctcttcgc
     1261 gtactgtttt tggcaacgtg gctctgccgc tggagctgga caacacaccg aaagacgagg
     1321 tcaaacgtcg cgtgacggaa ttgctgtcat tggttggtct tggcgataag catgatagct
     1381 acccgtcgaa tctttccggt gggcagaaac aacgtgtggc aattgcccgt gcgttagcca
     1441 gcaatcccaa agtattgctg tgtgatcaag ccaccagcgc gctggacccg gcaacgacac
     1501 gttctattct cgaactgctg aaagacatca accgccgtct ggggttgacg attctgttga
     1561 tcacccacga aatggacgtt gtgaagcgca tttgtgattg cgtggcggtc atcagcaatg
     1621 gagaactgat cgagcaggac acggtaagtg aagtgttctc gcatccgaaa acgccgctgg
     1681 cgcagaagtt tattcagtcg accctgcatc tggatatccc ggaagattac caggaacgtc
     1741 tgcaagcgga gccatttact gactgcgtgc cgatgctgcg tctggagttt accggtcaat
     1801 cggtcgatgc cccactgctt tctgaaaccg cgcgtcgttt caacgtcaac aacgcgatta
     1861 ttagcgcgca gatggattac gccggtggcg ttaagttcgg catcatgctg actgaaatgc
     1921 acggcacaca acaagatacg caagccgcca ttgcctggct gcaggaacac catgtaaaag
     1981 tagaggtact gggttatgtc tgactcgagg atccggctgc taacaaagcc cgaaaggaag
     2041 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     2101 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggatatccc gcaagaggcc
     2161 cggcagtacc ggcataacca agcctatgcc tacagcatcc agggtgacgg tgccgaggat
     2221 gacgatgagc gcattgttag atttcataca cggtgcctga ctgcgttagc aatttaactg
     2281 tgataaacta ccgcattaaa gcttatcgat gataagctgt caaacatgag aattcttgaa
     2341 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     2401 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     2461 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     2521 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     2581 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     2641 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     2701 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     2761 tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac
     2821 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     2881 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     2941 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     3001 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     3061 acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg
     3121 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     3181 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     3241 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     3301 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     3361 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     3421 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     3481 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     3541 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     3601 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     3661 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     3721 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     3781 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     3841 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     3901 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     3961 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     4021 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     4081 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     4141 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     4201 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     4261 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     4321 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     4381 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     4441 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     4501 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     4561 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     4621 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     4681 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     4741 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     4801 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctaac gatacgggtt
     4861 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     4921 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     4981 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     5041 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     5101 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     5161 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     5221 aggagcacga tcatgcgcac ccgtggccag gacccaacgc tgcccgagat gcgccgcgtg
     5281 cggctgctgg agatggcgga cgcgatggat atgttctgcc aagggttggt ttgcgcattc
     5341 acagttctcc gcaagaattg attggctcca attcttggag tggtgaatcc gttagcgagg
     5401 tgccgccggc ttccattcag gtcgaggtgg cccggctcca tgcaccgcga cgcaacgcgg
     5461 ggaggcagac aaggtatagg gcggcgccta caatccatgc caacccgttc catgtgctcg
     5521 ccgaggcggc ataaatcgcc gtgacgatca gcggtccaat gatcgaagtt aggctggtaa
     5581 gagccgcgag cgatccttga agctgtccct gatggtcgtc atctacctgc ctggacagca
     5641 tggcctgcaa cgcgggcatc ccgatgccgc cggaagcgag aagaatcata atggggaagg
     5701 ccatccagcc tcgcgtcgcg aacgccagca agacgtagcc cagcgcgtcg gccgccatgc
     5761 cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt
     5821 gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc
     5881 agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt
     5941 gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga
     6001 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
     6061 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     6121 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     6181 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc
     6241 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
     6301 tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc
     6361 actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc
     6421 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt
     6481 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc
     6541 tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca
     6601 gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc
     6661 acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca
     6721 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc
     6781 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc
     6841 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
     6901 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
     6961 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg
     7021 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca
     7081 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac
     7141 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc
     7201 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
     7261 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
     7321 gagcaccgcc gccgcaagga atggtgcatg ccgatcccgc gaaattaata cgactcacta
     7381 taggggaatt gtgagcggat aacaattccc ctctagaaat aat
//
