LOCUS       sequence-324827-        6540 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses MAT2A in E. coli BL21(DE3).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6540)
  AUTHORS   Firestone RS, Schramm VL
  TITLE     The Transition-State Structure for Human MAT2A from Isotope Effects.
  JOURNAL   J Am Chem Soc. 2017 Oct 4;139(39):13754-13760. doi: 
            10.1021/jacs.7b05803. Epub 2017 Sep 20.
  PUBMED    28880543
REFERENCE   2  (bases 1 to 6540)
  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"; doi: 
            "10.1021/jacs.7b05803"; journalName: "J Am Chem Soc"; date: 
            "2017-10-4- 4"; volume: "139"; issue: "39"; pages: "13754-13760"
COMMENT     Sequence Label: pET-MAT2A
FEATURES             Location/Qualifiers
     source          1..6540
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             92..109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             134..154
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1405..1422
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1475..1493)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1489..1536
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1573..2028
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1660..1679)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1870..1891
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2120..2935)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2120..2935)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2843..2862
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2927..2946
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3057..3645
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3546..3565
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3799..3816
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3831..3970
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3896..3915)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4056..4078
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4072..4263)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5072..6154)
                     /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     6116..6135
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6155..6232)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6438..6457
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcaccatcat catcatcatt cttctggtgt
      121 agatctgggt accgagaacc tgtacttcca atccatgaac ggacagctca acggcttcca
      181 tgaggcgttc atcgaggagg gcacattcct tttcacctca gagtcggtcg gggaaggcca
      241 cccagataag atttgtgacc aaatcagtga tgctgtcctt gatgcccacc ttcagcagga
      301 tcctgatgcc aaagtagctt gtgaaactgt tgctaaaact ggaatgatcc ttcttgctgg
      361 ggaaattaca tccagagctg ctgttgacta ccagaaagtg gttcgtgaag ctgttaaaca
      421 cattggatat gatgattctt ccaaaggttt tgactacaag acttgtaacg tgctggtagc
      481 cttggagcaa cagtcaccag atattgctca aggtgttcat cttgacagaa atgaagaaga
      541 cattggtgct ggagaccagg gcttaatgtt tggctatgcc actgatgaaa ctgaggagtg
      601 tatgccttta accattgtct tggcacacaa gctaaatgcc aaactggcag aactacgccg
      661 taatggcact ttgccttggt tacgccctga ttctaaaact caagttactg tgcagtatat
      721 gcaggatcga ggtgctgtgc ttcccatcag agtccacaca attgttatat ctgttcagca
      781 tgatgaagag gtttgtcttg atgaaatgag ggatgcccta aaggagaaag tcatcaaagc
      841 agttgtgcct gcgaaatacc ttgatgagga tacaatctac cacctacagc caagtggcag
      901 atttgttatt ggtgggcctc agggtgatgc tggtttgact ggacgcaaaa tcattgtgga
      961 cacttatggc ggttggggtg ctcatggagg aggtgccttt tcaggaaagg attataccaa
     1021 ggtcgaccgt tcagctgctt atgctgctcg ttgggtggca aaatcccttg ttaaaggagg
     1081 tctgtgccgg agggttcttg ttcaggtctc ttatgctatt ggagtttctc atccattatc
     1141 tatctccatt ttccattatg gtacctctca gaagagtgag agagagctat tagagattgt
     1201 gaagaagaat ttcgatctcc gccctggggt cattgtcagg gatctggatc tgaagaagcc
     1261 aatttatcag aggactgcag cctatggcca ctttggtagg gacagcttcc catgggaagt
     1321 gcccaaaaag cttaaatatt gacagtaaag gtggatacgg atccgaattc gagctccgtc
     1381 gacaagcttg cggccgcact cgagcaccac caccaccacc actgagatcc ggctgctaac
     1441 aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc
     1501 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
     1561 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     1621 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     1681 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     1741 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     1801 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     1861 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     1921 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     1981 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     2041 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     2101 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
     2161 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     2221 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     2281 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     2341 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
     2401 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
     2461 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
     2521 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     2581 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     2641 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     2701 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     2761 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     2821 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     2881 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac
     2941 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
     3001 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     3061 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     3121 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     3181 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     3241 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     3301 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     3361 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     3421 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     3481 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     3541 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     3601 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     3661 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     3721 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     3781 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     3841 tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa
     3901 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     3961 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     4021 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     4081 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     4141 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     4201 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     4261 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
     4321 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
     4381 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca
     4441 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc
     4501 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga
     4561 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca
     4621 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
     4681 ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat
     4741 gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc
     4801 ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct
     4861 ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag
     4921 ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg
     4981 gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg
     5041 agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     5101 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     5161 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
     5221 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
     5281 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
     5341 ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
     5401 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
     5461 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
     5521 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
     5581 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
     5641 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
     5701 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
     5761 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
     5821 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
     5881 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
     5941 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
     6001 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
     6061 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
     6121 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     6181 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     6241 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     6301 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     6361 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     6421 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     6481 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
//
