LOCUS       sequence-344823-        6912 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of MetN and MetI membrane proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6912)
  AUTHORS   Sharaf NG, Shahgholi M, Kim E, Lai JY, VanderVelde DG, Lee AT, Rees 
            DC
  TITLE     Characterization of the ABC methionine transporter from Neisseria 
            meningitidis reveals that lipidated MetQ is required for 
            interaction.
  JOURNAL   Elife. 2021 Aug 19;10. pii: 69742. doi: 10.7554/eLife.69742.
  PUBMED    34409939
REFERENCE   2  (bases 1 to 6912)
  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: "Elife."; 
            date: "2021-08-19"; volume: "10. pii: 69742. doi"; pages: " 
            10.7554/eLife.69742"
COMMENT     Sequence Label: NmMetNI-WT
FEATURES             Location/Qualifiers
     source          1..6912
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             16..38
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    53..77
                     /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)."
     promoter        complement(78..96)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      complement(138..185)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(996..1010)
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             complement(1026..1055)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             1068..1090
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1105..1129
                     /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)."
     promoter        complement(1130..1148)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1264..1341
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1342..2424
                     /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     complement(1361..1380)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3233..3424
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3418..3440)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3526..3665
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3581..3600
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3680..3697)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3851..4439)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3931..3950)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4610..5401,5402..5470))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5233..5252
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5471..5575)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5601..6056)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5738..5759)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5950..5969
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(6093..6140)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(6207..6224)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 gccatgagct ctatatctcc ttcttaaagt taaacaaaat tatttctaga ggggaattgt
       61 tatccgctca caattcccct atagtgagtc gtattaattt cgcgggatcg agatctggat
      121 atagttcctc ctttcagcaa aaaacccctc aagacccgtt tagaggcccc aaggggttat
      181 gctagttatt gctcagcggt ggcagcagcc aactcagctt cctttcgggc tttgttagca
      241 gccggatcct cgagtcagtc ctcccgaatc agttcgcgcc cgatgtcgga ttgggcgtgg
      301 atttggttgc cgcgtacttc gacgatttcg acgactttgc ctttatccaa gagggcggcg
      361 cggtcgcaca ggcggcggat gacgctcatt tcgtgggtta cgatgacgat ggttacgttg
      421 aagcgtttgt tgatgtcttc caaacattcc aagacgctgc gcgtggtggc ggggtcgagg
      481 gcggaagtgg gttcgtctgc gaggatgact tggggtttgg gcgcgagtgc gcgggcgatg
      541 ccgacacgtt gtttctgccc gccggaaagc tgggcgggat agtggccggc gcgttcggtc
      601 aagccgacga tttcaaggca ttctttaacg cgcgctttga ttttttcaga cggccatccg
      661 gcgatttcca aaggaaaggc aacattgtcg gcaacggtgc ggttgctcaa aagattaaac
      721 tgctgaaaca ccatgccgat attctgccga gcctgacgca atgcggcggc atcgagcgcg
      781 gtcagctctt gtccgcagac gttgaccttg ccgctgtcgg ggcgttccaa caggttaatc
      841 aggcgcaaca gggtggattt gcctgcaccc gaataaccca tcagcccgaa gatttcgccg
      901 tcgcggattt cgaggctggt cggctcgacg gcggcaaaac gggtcttgtc gcgcgtttgg
      961 taatgcttgg aaaccttgtc caaaataatc atatgcttgt cgtcgtcgtc gatatggccg
     1021 ctgctgtgat gatgatgatg atgatgatga tgatggccca tggtatatct ccttcttaaa
     1081 gttaaacaaa attatttcta gaggggaatt gttatccgct cacaattccc ctatagtgag
     1141 tcgtattaat ttcgcgggat cggcatgcac cattccttgc ggcggcggtg ctcaacggcc
     1201 tcaacctact actgggctgc ttcctaatgc aggagtcgca taagggagag cgtcgagatc
     1261 ccggacacca tcgaatggcg caaaaccttt cgcggtatgg catgatagcg cccggaagag
     1321 agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc
     1381 ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa
     1441 acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca
     1501 caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg
     1561 cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc
     1621 gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat
     1681 cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc
     1741 attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag
     1801 acacccatca acagtattat tttctcccat gaagacggta cgcgactggg cgtggagcat
     1861 ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg
     1921 gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat tcagccgata
     1981 gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat gcaaatgctg
     2041 aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc gctgggcgca
     2101 atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt agtgggatac
     2161 gacgataccg aagacagctc atgttatatc ccgccgttaa ccaccatcaa acaggatttt
     2221 cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg
     2281 aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat
     2341 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     2401 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc tcactcatta
     2461 ggcaccggga tctcgaccga tgcccttgag agccttcaac ccagtcagct ccttccggtg
     2521 ggcgcggggc atgactatcg tcgccgcact tatgactgtc ttctttatca tgcaactcgt
     2581 aggacaggtg ccggcagcgc tctgggtcat tttcggcgag gaccgctttc gctggagcgc
     2641 gacgatgatc ggcctgtcgc ttgcggtatt cggaatcttg cacgccctcg ctcaagcctt
     2701 cgtcactggt cccgccacca aacgtttcgg cgagaagcag gccattatcg ccggcatggc
     2761 ggccccacgg gtgcgcatga tcgtgctcct gtcgttgagg acccggctag gctggcgggg
     2821 ttgccttact ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg
     2881 ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta
     2941 aagtctggaa acgcggaagt cagcgccctg caccattatg ttccggatct gcatcgcagg
     3001 atgctgctgg ctaccctgtg gaacacctac atctgtatta acgaagcgct ggcattgacc
     3061 ctgagtgatt tttctctggt cccgccgcat ccataccgcc agttgtttac cctcacaacg
     3121 ttccagtaac cgggcatgtt catcatcagt aacccgtatc gtgagcatcc tctctcgttt
     3181 catcggtatc attaccccca tgaacagaaa tcccccttac acggaggcat cagtgaccaa
     3241 acaggaaaaa accgccctta acatggcccg ctttatcaga agccagacat taacgcttct
     3301 ggagaaactc aacgagctgg acgcggatga acaggcagac atctgtgaat cgcttcacga
     3361 ccacgctgat gagctttacc gcagctgcct cgcgcgtttc ggtgatgacg gtgaaaacct
     3421 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag
     3481 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca
     3541 gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga gcagattgta
     3601 ctgagagtgc accattgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca
     3661 tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     3721 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     3781 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     3841 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     3901 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     3961 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     4021 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     4081 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     4141 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4201 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     4261 agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga
     4321 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     4381 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     4441 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     4501 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     4561 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     4621 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     4681 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     4741 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     4801 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     4861 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     4921 ttgctgcagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     4981 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     5041 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     5101 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     5161 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     5221 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     5281 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     5341 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     5401 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     5461 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     5521 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     5581 ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     5641 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     5701 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     5761 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     5821 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     5881 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     5941 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     6001 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca
     6061 ttcgccaatc cggatatagt tcctcctttc agcaaaaaac ccctcaagac ccgtttagag
     6121 gccccaaggg gttatgctag ttattgctca gcggtggcag cagccaactc agcttccttt
     6181 cgggctttgt tagcagccgg atctcagtgg tggtggtggt ggtgggtacc tcaacgtttg
     6241 tcgagtttcc gcgccaacgc gttgccggtg ctttgaatca ggatgaccag cagcacgagg
     6301 agggcgacga tgaagatgat gacttcggtt tggtagcggt agtagccgta gcggatggcg
     6361 aggtcgccca agccgccgcc gcctatcatc cctgccgccg cgctgtatga caaaagcccg
     6421 atggcaagca cggtaatgct ggaaaccatg cccgcgcgcg cttcgttcaa gaggactttg
     6481 cagacgatgg caatcggcgg cgcacccatc gcggcggcgg cttcaattac gcctttgggg
     6541 acttcgcgca ggttttgttc caccagtcgg gcaaaataaa acaatcccga cacgctcaac
     6601 accagcgagg cggcaaccgg accgatggtg ctgccgacga tggcgcgtgt ggcgggtatc
     6661 atcgcaatca tcaggatgac gaaggggaag gcgcgcatga ggttgacgag gttgtcgagc
     6721 aggaagttca ccagcttgtt gtaatgcagt tggcggctgg aggttacgaa gagcagcacg
     6781 cccagcagcg tgccgaagat gacggcgaat gtggtggaca agccgaccat cacgaaggtt
     6841 tcgcccaagg cgcggaagat ttcgtctttc atgccgacga tggtggaaac ggcttgttgg
     6901 aatgttaagt ct
//
