LOCUS       sequence-379186-        9433 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses NG-tagged ANME2cLM014 MCR in Methanococcus maripaludis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9433)
  AUTHORS   Shao N, Fan Y, Chou CW, Yavari S, Williams RV, Amster IJ, Brown SM, 
            Drake IJ, Duin EC, Whitman WB, Liu Y
  TITLE     Expression of divergent methyl/alkyl coenzyme M reductases from 
            uncultured archaea.
  JOURNAL   Commun Biol. 2022 Oct 20;5(1):1113. doi: 10.1038/s42003-022-04057-6.
  PUBMED    36266535
REFERENCE   2  (bases 1 to 9433)
  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: "Commun 
            Biol."; date: "2022-10-20"; volume: "5(1):1113. doi"; pages: " 
            10.1038/s42003-022-04057-6"
COMMENT     Sequence Label: Ppst93-ANME2cLM014 MCR-NG
FEATURES             Location/Qualifiers
     source          1..9433
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2694..2717
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             join(2727..2750,2751..2786,2787..2810)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="WSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     primer_bind     complement(6653..6672)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6772..6794
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6832..6850)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6918..7022
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7023..7091,7092..7883)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(7241..7260)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8054..8642
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8543..8562
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 actgtatgct tttttcattt cctttatttt gtcctttcct ttctttgtta tctggtaata
       61 tccttccttc tttcctgcat acttcaaaaa tccaaggttt tttaagttat cgagataaga
      121 attactagat gaattcgcgc ccggttctag atttattcat aaaataggaa ctaaaatacc
      181 aaaatagaaa catttatata catttgatgt gtagggtata ccataaacct gggaggtgtc
      241 tcatatggca gataaaattg atctttacga tgatagagga aaacttctta aatcagatgt
      301 tgatttacaa gcagtttcac cattaaaaaa ctcagcaatt cttaacatgg ttaacacagt
      361 taaaagaact gttgcagtta accttgcagg aattgaaaaa gcatgtaaaa acgcaggata
      421 cgcaggacag atgagacaca ttccaggaag agaagttgat attgatccaa cagcaaaagc
      481 agataaaatt gcagcaaaag ttaaagaatt agttcaagtt gaaaaaggag atgatacaga
      541 tgttactgtt ttaggtggag gaaaatttct tagagttcag gttccaacaa gaagaattga
      601 agcaggagca gaatatgttg caggaatgac ttgtgcagca gcagcagttg ttgaagcact
      661 tagagaagaa tataaccttg gactttatga tactccatac gttaaatcag cagtttgggg
      721 aacataccca caaactatgg atatgaaagg tggaaacgtt ttatcagttc tttcaattcc
      781 acagaatgat gaaggacttg gatttgcact tagaaacatt atggcaaacc accttgcaat
      841 gctttcacaa agaaacgcaa tgaactgtgc agcaatttca tcaattttag aaaactgtgg
      901 agtttttgaa atgggacagg cacttggact ttttgaaaga taccaacttt tagcaatggc
      961 ataccaggga cttaacgcaa acaacatggt ttacgatatg gttaaaaaca acggaaaaac
     1021 aggaactatt ggaacagttg ttcaagaaac tgttgaaaga gcattagatg atggaattat
     1081 tgcagttgat aaaacaatgc catcaggata caaagtttac aaaactaacg atgtttgtct
     1141 ttggaacgca tattgtgcag caggaacaat ggcagcaact atggttaatt gtggagcatt
     1201 aagaggagca caggcagttt catcaacact tctttacttt aacgatatga ttgaaaaaga
     1261 aacttcatta ccaggatgtg attttggaag agttgaagga acagcagttg gattttcatt
     1321 tttctcacac tcaatttatg gaggcggagg accaggagtt tttaatggaa atcacgttgt
     1381 tacaagacac tcaactggaa tggcaattcc atgtgttgca gcagcagttg cattagatgc
     1441 aggaactcaa atgttttcac cagaatcaac atcagcatta gttttagata cttacaaaga
     1501 tgtgccaatc atgatgcagc cattaaagga ggtcgcagca gcggtgtaaa gaggaattgt
     1561 tttatggtta caactcaaga atcagatcca gttcagtacg aaatttttcc acaaagaatg
     1621 cttaacccag caacagcaca ggaacttctt aacagattag aacaaattgg aggaattatt
     1681 agagttttag ttcagggacc aagattacca ttaactgttc catacggacc aggaaaagga
     1741 gttccagttg atcacccaga tagagaaatt gttagagttg gagataaagt tatggaactt
     1801 cacgttaaag ttggaagaat tagattagaa tcagaagaag atgttatgga agaaattaga
     1861 gaaatttgtg aagaagttct tccatttcca tttgaactta aagaaggatt atttcttaga
     1921 agaaaagcaa cagttactga ttacgcaaaa tacggaccag atgaattagt tgaagataaa
     1981 agaatttacg gaatgtcaga tccaaacatg aagccgaccg cgtctattgt gtgcgatcca
     2041 cgagaggatt aagaaaaatg tttggaagag aaactcaagt tattgattgt agacagggaa
     2101 tgggaccagg aagaggtgga ggattagcac aaagaggaac attatcagaa actggaagac
     2161 cagatgttat tgcaattgca atgacaccag gaagaagaca cattactaaa ccagtttgtg
     2221 aaattacaca cggattaaga agagaaggaa ttcaggtttc agttttagtt ttagaagcag
     2281 gagcaggagt tccagaagat gcaccaggag atattatggc aaaaggatac ggaccaaaat
     2341 ttggacttac tcaaaaagaa attgaacaga ttgaaaaaca ccacgcagtt cttattcacc
     2401 ttggaaacat tggatcacac gttatttaca aaacaaaata cattcttaaa tatgttgatg
     2461 ttccagcaat tattgtttgt caaaacccag ttgattttga agattttgca agagcaggaa
     2521 ttaaaacaag attagttatg ccaaaaaaca gagatattga aacaagagga actgttatgg
     2581 aaattgttac aggaattact agaggagtta cttgtcaaag aaacagactt aacgaaatcg
     2641 taaaggcaac tataaaaaca ataccacaat atacataagg agaaatagtt atggattaca
     2701 aagatgatga tgataaaggc gccagctggt cacacccaca atttgaaaaa ggaggtggat
     2761 caggaggtgg atcaggaggt tcagcatggt cacatcctca gtttgaaaaa tcagcaatgg
     2821 catataaacc acaatattac ccaggatcat catcagttgc agttaataga agaaaacaca
     2881 tgtcaggaga tattgaaaaa cttagaacag tttcagatga tgatttagtt gcagcattag
     2941 gacacagagc accaggagca gattatccat caacacaccc accattagca gaaatgggag
     3001 aaccagattg tccaattaga caattagttg aaccaactcc aggagcagca gcaggagata
     3061 gagttagata ctcacagttt acagattcaa tgtattcagc accatcaact ccatacttta
     3121 gatcatacta cgcagcaatt aatcacagag gagttgatcc aggaacttta tcaggaagac
     3181 agattgttga agcaagagaa agagatatgg aagtttacac aaaagcaact attgaatcag
     3241 aaatgacatg tccaggatta gcaggattaa gaggatgtac tgttcacgga cactcactta
     3301 gattagatga agatggaaca atgtttgata tgcttcaaag agttcactta gaaggtggaa
     3361 acattattga agataaagat caggttggag ttccaattga tagaaaagtt aaccttggaa
     3421 aaccaatgac tgaagcagaa gcaaagaaaa gaacaactat ttacagaaca gatggagtta
     3481 gatacagaac tgaagaagaa gttttagatc acgttcactt agttcaccac agaagaacaa
     3541 tgtatggatt tagaccagaa aatgcatcag agaccgcggc aggtaccggg gacatcaaat
     3601 accacacggt gtaaaggagg tagatacaaa tgacatacaa atacccatca gagaaaatat
     3661 tcgttgaagc gctgagagag aagtttgcag gattagatct ttcagaacag aaagttaaat
     3721 acgttagagc aggatactta caatcagcaa gaaaaagaga atttcaggca gcaggagaaa
     3781 gagttgcaga aaaacgagga attaaacaat acgatgcaaa cgttcacctt ggaggaatga
     3841 ctttaggaca aagacagtta gttccataca aactttcaac aagaccagat attgttgaag
     3901 gagatgattt acactacgtt aataatccag caatgcaaca gatgtgggat gatatgaaaa
     3961 gaactattat tgttggaatg gatttagcac acgaaacatt agaaaaacga cttggaaaag
     4021 aagttacacc agaaactatt aatggataca tggaagcagt taatcacact atgccaggag
     4081 cagcaattgt tcaggaacac atggttgaaa cacacccagg attagttgat gattgttacg
     4141 ttaaaatttt tactggagat gatgaattag cagatgaaat tgatccacaa tacgttatta
     4201 acattaacaa actttttgat aaagaagatc agaacgaaaa acttaaagca gcaattggaa
     4261 aaacaacttg gcaagcagtt cacattccaa ctattgttgt tagatcatgt gatggaggaa
     4321 atacatcaag atggtcagca atgcaaattg gaatgtcatt tattgcagca tacaacatgt
     4381 gtgcaggaga agcagcagtt gcagatttag catttgcagc aaaacacgca gcagcaattc
     4441 agatggcaga aatgttacca gcaagaagag caagatcacc aaatgaacct ggaggattat
     4501 catttggata ttgtgcagat atggttcaaa caatgagaat taaaccagaa gatccagttt
     4561 ggtacacttt agaagttgtt gcatgtggaa caatgttata tgatcagatt tggttaggat
     4621 catacatgtc tggaggagtt ggatttacac aatatgcaac tgcagcatac acaaacgatg
     4681 ttttagatga ttttacatac tacggatacg attacgcact taacaaatac ggaccagatg
     4741 gaactgcacc aaatgattta gcaacagcaa ctgatattgc aacagaaaca actcttaacg
     4801 gaatggaatc atacgaagat tacccaactt tattagaaga tcactttgga ggatcacaga
     4861 gagcaggaat tttagcagca gcatcagcat gtgcaactgg aattgcaaca ggaaatgcac
     4921 aagttgcatt atcaggatgg tatatgtcaa tgtacgttca caaagaagga tggggaagat
     4981 taggattttt cggatacgat ttacaagatc agtgtggagc aacaaatgtt tgttcatacc
     5041 agggagatga aggagaatgt ttagaactta gaggagcaaa ctacccaaat tacgcaatga
     5101 atgttggaca ccaaggagaa tatgcaggat ttactggagc agcacacgca ggagcacacg
     5161 atgcatactg ttgtaatcca ttagttaaag tttgttttgc agatccatca ttagtttttg
     5221 atttttcata cattagaaga gaatacgcaa aaggtggaat gagaactttt agaccagcag
     5281 gagaaagatc attagttatt ccagcaggaa tttaaactag tagcggccgc tgcaggatat
     5341 agcaaaagtg ggacttaagt tcccactttt gctattttat aaaaaacgcc ctattcgtaa
     5401 tatactattc aaaactaaat attcattgaa atatttgggt aataattaat attaatctaa
     5461 tatttagata ttgagatata ataataacat ttttttaaaa aatcatctat tttcatctta
     5521 attttatata ttaaagccca ttttttgagt attcaaattc aaattattgt gttattaaca
     5581 tcttatatat aaacttttct atttaatgtt aatgaaaaag tgaatatata tacatagagt
     5641 aatgttatga tgtatatatc aaaaaaaact agaggagacg accttccatg acagaatata
     5701 aaccaacagt tagattagca acaagagatg acgtacctag ggcagtaaga actcttgcag
     5761 cagcttttgc agattatcca gcaacaagac acacagttga tccagataga cacattgaaa
     5821 gagttacaga attacaagaa ttatttttaa caagagttgg tcttgatata ggtaaagtat
     5881 gggttgcaga tgatggagca gcagttgcag tttggacaac accagaatca gttgaagctg
     5941 gtgctgtttt tgcagaaatt ggaccaagaa tggctgaatt atcaggatca agattagcag
     6001 ctcagcaaca gatggaagga ttacttgcac cacacagacc aaaagaacca gcatggtttt
     6061 tagcaacagt tggagtttca ccagatcacc agggaaaagg tttaggttct gctgttgtac
     6121 ttcctggtgt tgaagcagct gaaagagcag gagttccagc atttttagaa acatcagcac
     6181 caagaaatct tccattttat gaaagacttg gatttacagt tacagcagat gttgaagttc
     6241 cagaaggacc aagaacatgg tgtatgacaa gaaaaccagg agcataacca tggctccgac
     6301 cgaagccacc cggggcggcc ccgccgaccc cgcacccgcc cccgaggccc accgcggggg
     6361 acacaccgaa cacgccgacc ctgctgaaca cgcggcgcag ttcggtgccc aggagcggat
     6421 cgggaattaa ttcgaagctg ctggtgaaag agaccctatc ttacctgcta aaatctaagt
     6481 taattactaa tttattatta atttattatt agattgggta aaatagtaaa agaaaactaa
     6541 aggaaaccta atatggtttc ctttttttat atatttttaa ttcactgggg ggatttaaaa
     6601 cttcattttt aatttaaaag gatctaggtg aagatccttg gtgcactctc agtacaatct
     6661 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     6721 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     6781 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     6841 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     6901 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     6961 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     7021 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     7081 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     7141 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     7201 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     7261 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     7321 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     7381 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     7441 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     7501 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     7561 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     7621 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     7681 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     7741 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     7801 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     7861 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     7921 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     7981 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     8041 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     8101 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     8161 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     8221 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     8281 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     8341 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     8401 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     8461 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     8521 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     8581 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     8641 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     8701 tggtacccat tatttagttt ttttttatca ccacaacgta taaaccccct ttcagaagaa
     8761 aacagagaat gaaaacagat tttaatttat atatcgtatc tataatttag ttttttttta
     8821 ttgcagtttt agaagtgcgt ttgtcctgga gcgaaataca cgatatcgtg tagtctcctt
     8881 cagatgaaaa taatattttt aattttcaga aatattactg gaaaaatacc tgaaaaatat
     8941 ttgaaaaaaa aacatacacc cagcggggaa aaatcgaaca ttttgaaaaa gtctatttat
     9001 agggtttaaa cagggccaag acctgcggga tataccccaa aaatgttcga taaaaaaatc
     9061 gaacattttt cgaacatttt ttataatgtt cgatgtgtgc gagtatgttc gataaaaaaa
     9121 tcgaacattt tgaaagacaa tttgaagaga tataaaaaaa ataatctatt ataatataga
     9181 tatataatat gttcgatatg ttcgatattt ttttatatat tatattatat ttaaccttgt
     9241 attgattatt gaataatatt tattattact ttttttgatt tcttgtaaaa aatcgaacat
     9301 attttacata ttgtaaaaaa taaaaaagat ttaaaaaaag tgtccgaaaa agttaaacaa
     9361 acattattat taaaatgtta gtttaatatg tatcttgata tgctcttctt tctttcatgc
     9421 ttaaacttgt ata
//
