LOCUS       sequence-342851-        6072 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid with a TEV removable N-term MBP tag, a TEV 
            removable C-term 8xHis tag, and Chloramphenicol resistance. NikJ 
            C199U cloned in the cloning site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6072)
  AUTHORS   Caceres JC, Bailey CA, Yokoyama K, Greene BL
  TITLE     Selenocysteine substitutions in thiyl radical enzymes.
  JOURNAL   Methods Enzymol. 2022;662:119-141. doi: 10.1016/bs.mie.2021.10.014. 
            Epub 2021 Dec 7.
  PUBMED    35101207
REFERENCE   2  (bases 1 to 6072)
  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: "Methods 
            Enzymol."; date: "2022"; volume: "662:119-141. doi"; pages: " 
            10.1016/bs.mie.2021.10.014. Epub 2021 Dec 7"
COMMENT     Sequence Label: pCm N-term MBP NikJC199U
FEATURES             Location/Qualifiers
     source          1..6072
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(119..1201)
                     /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     1163..1182
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1202..1279)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1325..1344
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1325..1343
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1344..1368
                     /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)."
     CDS             1412..2512
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      2369..2378
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     2486..2509
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2561..2572
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             2585..2605
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3983..4003
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4016..4039
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(4092..4110)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4106..4153
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(4277..4296)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4468..4570
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             4571..5230
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(4637..4656)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      complement(5246..5995)
                     /direction=LEFT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     primer_bind     complement(5256..5273)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(5496..5515)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 catgttagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
       61 catcggtcga gatcccggtg cctaatgagt gagctaagtt acattaattg cgttgcgctc
      121 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
      181 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgata
      241 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
      301 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
      361 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat gtccgcacca acgcgcagcc
      421 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
      481 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
      541 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
      601 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
      661 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
      721 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
      781 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
      841 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
      901 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
      961 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     1021 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     1081 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     1141 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     1201 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     1261 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
     1321 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tgtagaaata
     1381 attttgttta actttaataa ggagatatac catgaaaatc gaagaaggta aactggtaat
     1441 ctggattaac ggcgataaag gctataacgg tctcgctgaa gtcggtaaga aattcgagaa
     1501 agataccgga attaaagtca ccgttgagca tccggataaa ctggaagaga aattcccaca
     1561 ggttgcggca actggcgatg gccctgacat tatcttctgg gcacacgacc gctttggtgg
     1621 ctacgctcaa tctggcctgt tggctgaaat caccccggac aaagcgttcc aggacaagct
     1681 gtatccgttt acctgggatg ccgtacgtta caacggcaag ctgattgctt acccgatcgc
     1741 tgttgaagcg ttatcgctga tttataacaa agatctgctg ccgaacccgc caaaaacctg
     1801 ggaagagatc ccggcgctgg ataaagaact gaaagcgaaa ggtaagagcg cgctgatgtt
     1861 caacctgcaa gaaccgtact tcacctggcc gctgattgct gctgacgggg gttatgcgtt
     1921 caagtatgaa aacggcaagt acgacattaa agacgtgggc gtggataacg ctggcgcgaa
     1981 agcgggtctg accttcctgg ttgacctgat taaaaacaaa cacatgaatg cagacaccga
     2041 ttactccatc gcagaagctg cctttaataa aggcgaaaca gcgatgacca tcaacggccc
     2101 gtgggcatgg tccaacatcg acaccagcaa agtgaattat ggtgtaacgg tactgccgac
     2161 cttcaagggt caaccatcca aaccgttcgt tggcgtgctg agcgcaggta ttaacgccgc
     2221 cagtccgaac aaagagctgg caaaagagtt cctcgaaaac tatctgctga ctgatgaagg
     2281 tctggaagcg gttaataaag acaaaccgct gggtgccgta gcgctgaagt cttacgagga
     2341 agagttggcg aaagatccac gtattgccgc caccatggaa aacgcccaga aaggtgaaat
     2401 catgccgaac atcccgcaga tgtccgcttt ctggtatgcc gtgcgtactg cggtgatcaa
     2461 cgccgccagc ggtcgtcaga ctgtcgatga agccctgaaa gacgcgcaga ctaattcgag
     2521 ctcgaacaac aacaacaata acaataacaa caacctcggg atcgagggaa ggatttcaga
     2581 attcgaaaac ctgtattttc aggggatggg cgaaatcgtg gacgaaccgc gtaaaatcct
     2641 ggtcatctgg ccgccgcaag tgctgtcata cttcaatgct ggtcatcacc tggcactgta
     2701 tcaggtgacc ggtcatctgc gtgctgtgta cccgggtgcg caagttaccg ccattgatgc
     2761 atcagttgaa cgtctgacgt ggaaagatct gggttcgcgc ctgttccagg aacgttatga
     2821 tgtgattgcc gttatgaatg aatttgatgg catcgacggt ctgcgtcgcc tggttagcta
     2881 cgcccgtgca ctgaacccgg atgcaccgat tgttaccttt ggccgtctgt ctggtgtcaa
     2941 tccgcgcttt ttcgaacgtt ttgatattga cgccatcgtg gaaaacggcg atttcgaaag
     3001 cggtgtggtt gcggccatcg aagcctttcg cggccgtgca gctgatgcac cgggtgttca
     3061 tctgcgtcgc gacggttcgt gggcagcacc gaaacgtcgc ggtgatctgc tgccggcaga
     3121 agactggtat ctgccgaccc cggatgaaat tccgtatggt cactacgacg cactgtactt
     3181 tgatgacgct aacaagttct agggtatccc gcgtcgccgt gaactggtcg tgccggcagc
     3241 tcgtggttgt ccggtcggtt gcagctattg tgaagtgccg ctgatttaca cgcgcaaaga
     3301 acgccgtctg accgttgaac gtacgattca gtatatcgaa gaatcttttg cggccgcacc
     3361 gttcgaatat gtcgcttttt acgcgccgac cttcacgctg gataaaaagt ggatggaaga
     3421 actgtgccgc gaactgaccg gccgtggttc accgtatccg tggaagtgtg cgaccacggt
     3481 tcatcacctg agcgagggta tgattcgctc tatgggcgct gcgggttgcg ttcgtatcag
     3541 tgtcggtctg gaaacgctgg atccgcagga aggcgcactg ctgccgcgtg caaaacaaaa
     3601 gtccgctatg gatctggacc gtctgagtgc ctggtgcaaa gaatccggta ttgaactgaa
     3661 ttgttttgtt atcgtcggcc tgccgggtac cacgattgaa ggcgcggaac gcaccatcga
     3721 agcagtgcag gaacgtggcg gtcgtgctcg tgcaacggtt tatgcaccgt gggaactggc
     3781 aggtccggat ctgctggacc atgaaatggc cgcatttaat cgccaactgt tcgtcccggg
     3841 cacccacgca ctgacggatg acgaactggc tcgtggctac gatattgtgt ttggttccca
     3901 gccggacgtc acccaagtgt tcgaaaacat cccggcgcgc gctgcgggcc cggccctgcg
     3961 tccggcacag ggaaacggta ccgaaaacct gtattttcag ggctcgacaa tggatcacca
     4021 ccaccaccac caccaccact aacctaggct gctaaacaaa gcccgaaagg aagctgagtt
     4081 ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
     4141 gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt
     4201 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     4261 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     4321 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     4381 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     4441 tagacggttt ttcgcccttt gacgttgtga tcggcacgta agaggttcca actttcacca
     4501 taatgaaata agatcactac cgggcgtatt ttttgagtta tcgagatttt caggagctaa
     4561 ggaagctaaa atggagaaaa aaatcactgg atataccacc gttgatatat cccaatggca
     4621 tcgtaaagaa cattttgagg catttcagtc agttgctcaa tgtacctata accagaccgt
     4681 tcagctggat attacggcct ttttaaagac cgtaaagaaa aataagcaca agttttatcc
     4741 ggcctttatt cacattcttg cccgcctgat gaatgctcat ccggaatttc gtatggcaat
     4801 gaaagacggt gagctggtga tatgggatag tgttcaccct tgttacaccg ttttccatga
     4861 gcaaactgaa acgttttcat cgctctggag tgaataccac gacgatttcc ggcagtttct
     4921 acacatatat tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg
     4981 gtttattgag aatatgtttt tcgtctcagc caatccctgg gtgagtttca ccagttttga
     5041 tttaaacgtg gccaatatgg acaacttctt cgcccccgtt ttcaccatgg gcaaatatta
     5101 tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag gttcatcatg ccgtttgtga
     5161 tggcttccat gtcggcagaa tgcttaatga attacaacag tactgcgatg agtggcaggg
     5221 cggggcgtaa gcagcatgca gcgctcttcc gcttcctcgc tcactgactc gctacgctcg
     5281 gtcgttcgac tgcggcgagc ggtgtcagct cactcaaaag cggtaatacg gttatccaca
     5341 gaatcagggg ataaagccgg aaagaacatg tgagcaaaaa gcaaagcacc ggaagaagcc
     5401 aacgccgcag gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     5461 ctcaagccag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     5521 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     5581 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgttggtatc tcagttcggt
     5641 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     5701 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     5761 ggcagcagcc attggtaact gatttagagg actttgtctt gaagttatgc acctgttaag
     5821 gctaaactga aagaacagat tttggtgagt gcggtcctcc aacccactta ccttggttca
     5881 aagagttggt agctcagcga accttgagaa aaccaccgtt ggtagcggtg gtttttcttt
     5941 atttatgaga tgatgaatca atcggtctat caagtcaacg aacagctatt ccgttactct
     6001 agatttcagt gcaatttatc tcttcaaatg tagcacctga agtcagcccc atacgatata
     6061 agttgtaatt ct
//
