LOCUS       sequence-342848-        6043 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid with a TEV removable C-term 8xHis tagged MBP 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 6043)
  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 6043)
  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 C-term MBP NikJC199U
FEATURES             Location/Qualifiers
     source          1..6043
                     /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             2789..2809
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2810..3910
                     /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      3767..3776
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     3884..3907
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             3972..3995
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(4048..4066)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4062..4109
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(4233..4252)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4439..4541
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             4542..5201
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(4608..4627)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      complement(5217..5966)
                     /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(5227..5244)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(5467..5486)
                     /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 accagtgaga
      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 catgggcgaa atcgtggacg aaccgcgtaa
     1441 aatcctggtc atctggccgc cgcaagtgct gtcatacttc aatgctggtc atcacctggc
     1501 actgtatcag gtgaccggtc atctgcgtgc tgtgtacccg ggtgcgcaag ttaccgccat
     1561 tgatgcatca gttgaacgtc tgacgtggaa agatctgggt tcgcgcctgt tccaggaacg
     1621 ttatgatgtg attgccgtta tgaatgaatt tgatggcatc gacggtctgc gtcgcctggt
     1681 tagctacgcc cgtgcactga acccggatgc accgattgtt acctttggcc gtctgtctgg
     1741 tgtcaatccg cgctttttcg aacgttttga tattgacgcc atcgtggaaa acggcgattt
     1801 cgaaagcggt gtggttgcgg ccatcgaagc ctttcgcggc cgtgcagctg atgcaccggg
     1861 tgttcatctg cgtcgcgacg gttcgtgggc agcaccgaaa cgtcgcggtg atctgctgcc
     1921 ggcagaagac tggtatctgc cgaccccgga tgaaattccg tatggtcact acgacgcact
     1981 gtactttgat gacgctaaca agttctaggg tatcccgcgt cgccgtgaac tggtcgtgcc
     2041 ggcagctcgt ggttgtccgg tcggttgcag ctattgtgaa gtgccgctga tttacacgcg
     2101 caaagaacgc cgtctgaccg ttgaacgtac gattcagtat atcgaagaat cttttgcggc
     2161 cgcaccgttc gaatatgtcg ctttttacgc gccgaccttc acgctggata aaaagtggat
     2221 ggaagaactg tgccgcgaac tgaccggccg tggttcaccg tatccgtgga agtgtgcgac
     2281 cacggttcat cacctgagcg agggtatgat tcgctctatg ggcgctgcgg gttgcgttcg
     2341 tatcagtgtc ggtctggaaa cgctggatcc gcaggaaggc gcactgctgc cgcgtgcaaa
     2401 acaaaagtcc gctatggatc tggaccgtct gagtgcctgg tgcaaagaat ccggtattga
     2461 actgaattgt tttgttatcg tcggcctgcc gggtaccacg attgaaggcg cggaacgcac
     2521 catcgaagca gtgcaggaac gtggcggtcg tgctcgtgca acggtttatg caccgtggga
     2581 actggcaggt ccggatctgc tggaccatga aatggccgca tttaatcgcc aactgttcgt
     2641 cccgggcacc cacgcactga cggatgacga actggctcgt ggctacgata ttgtgtttgg
     2701 ttcccagccg gacgtcaccc aagtgttcga aaacatcccg gcgcgcgctg cgggcccggc
     2761 cctgcgtccg gcacagggaa acggtaccga aaacctgtat tttcagggca tgaaaatcga
     2821 agaaggtaaa ctggtaatct ggattaacgg cgataaaggc tataacggtc tcgctgaagt
     2881 cggtaagaaa ttcgagaaag ataccggaat taaagtcacc gttgagcatc cggataaact
     2941 ggaagagaaa ttcccacagg ttgcggcaac tggcgatggc cctgacatta tcttctgggc
     3001 acacgaccgc tttggtggct acgctcaatc tggcctgttg gctgaaatca ccccggacaa
     3061 agcgttccag gacaagctgt atccgtttac ctgggatgcc gtacgttaca acggcaagct
     3121 gattgcttac ccgatcgctg ttgaagcgtt atcgctgatt tataacaaag atctgctgcc
     3181 gaacccgcca aaaacctggg aagagatccc ggcgctggat aaagaactga aagcgaaagg
     3241 taagagcgcg ctgatgttca acctgcaaga accgtacttc acctggccgc tgattgctgc
     3301 tgacgggggt tatgcgttca agtatgaaaa cggcaagtac gacattaaag acgtgggcgt
     3361 ggataacgct ggcgcgaaag cgggtctgac cttcctggtt gacctgatta aaaacaaaca
     3421 catgaatgca gacaccgatt actccatcgc agaagctgcc tttaataaag gcgaaacagc
     3481 gatgaccatc aacggcccgt gggcatggtc caacatcgac accagcaaag tgaattatgg
     3541 tgtaacggta ctgccgacct tcaagggtca accatccaaa ccgttcgttg gcgtgctgag
     3601 cgcaggtatt aacgccgcca gtccgaacaa agagctggca aaagagttcc tcgaaaacta
     3661 tctgctgact gatgaaggtc tggaagcggt taataaagac aaaccgctgg gtgccgtagc
     3721 gctgaagtct tacgaggaag agttggcgaa agatccacgt attgccgcca ccatggaaaa
     3781 cgcccagaaa ggtgaaatca tgccgaacat cccgcagatg tccgctttct ggtatgccgt
     3841 gcgtactgcg gtgatcaacg ccgccagcgg tcgtcagact gtcgatgaag ccctgaaaga
     3901 cgcgcagact aattcgagct cgaacaacaa caacaataac aataacaaac aacctcgggt
     3961 cgacaatgga tcaccaccac caccaccacc accactaacc taggctgcta aacaaagccc
     4021 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     4081 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga
     4141 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     4201 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     4261 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     4321 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     4381 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttcttttg
     4441 atcggcacgt aagaggttcc aactttcacc ataatgaaat aagatcacta ccgggcgtat
     4501 tttttgagtt atcgagattt tcaggagcta aggaagctaa aatggagaaa aaaatcactg
     4561 gatataccac cgttgatata tcccaatggc atcgtaaaga acattttgag gcatttcagt
     4621 cagttgctca atgtacctat aaccagaccg ttcagctgga tattacggcc tttttaaaga
     4681 ccgtaaagaa aaataagcac aagttttatc cggcctttat tcacattctt gcccgcctga
     4741 tgaatgctca tccggaattt cgtatggcaa tgaaagacgg tgagctggtg atatgggata
     4801 gtgttcaccc ttgttacacc gttttccatg agcaaactga aacgttttca tcgctctgga
     4861 gtgaatacca cgacgatttc cggcagtttc tacacatata ttcgcaagat gtggcgtgtt
     4921 acggtgaaaa cctggcctat ttccctaaag ggtttattga gaatatgttt ttcgtctcag
     4981 ccaatccctg ggtgagtttc accagttttg atttaaacgt ggccaatatg gacaacttct
     5041 tcgcccccgt tttcaccatg ggcaaatatt atacgcaagg cgacaaggtg ctgatgccgc
     5101 tggcgattca ggttcatcat gccgtttgtg atggcttcca tgtcggcaga atgcttaatg
     5161 aattacaaca gtactgcgat gagtggcagg gcggggcgta agcagcatgc agcgctcttc
     5221 cgcttcctcg ctcactgact cgctacgctc ggtcgttcga ctgcggcgag cggtgtcagc
     5281 tcactcaaaa gcggtaatac ggttatccac agaatcaggg gataaagccg gaaagaacat
     5341 gtgagcaaaa agcaaagcac cggaagaagc caacgccgca ggcgtttttc cataggctcc
     5401 gcccccctga cgagcatcac aaaaatcgac gctcaagcca gaggtggcga aacccgacag
     5461 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5521 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5581 atagctcacg ctgttggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5641 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5701 ccaacccggt aagacacgac ttatcgccac tggcagcagc cattggtaac tgatttagag
     5761 gactttgtct tgaagttatg cacctgttaa ggctaaactg aaagaacaga ttttggtgag
     5821 tgcggtcctc caacccactt accttggttc aaagagttgg tagctcagcg aaccttgaga
     5881 aaaccaccgt tggtagcggt ggtttttctt tatttatgag atgatgaatc aatcggtcta
     5941 tcaagtcaac gaacagctat tccgttactc tagatttcag tgcaatttat ctcttcaaat
     6001 gtagcacctg aagtcagccc catacgatat aagttgtaat tct
//
