LOCUS       sequence-342843-        6330 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid with a ULP1 removable N-term MBP SUMO 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 6330)
  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 6330)
  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 SMT3 NikJC199U
FEATURES             Location/Qualifiers
     source          1..6330
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      686..1435
                     /direction=RIGHT
                     /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     1166..1185
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1408..1425
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(1451..2110)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2025..2044
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2111..2213)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     2385..2404
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(2528..2575)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2571..2589
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2642..2665)
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             complement(2678..2698)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(4082..4372)
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             complement(4427..5527)
                     /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"
     primer_bind     complement(4430..4453)
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     regulatory      4561..4570
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    5571..5595
                     /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)."
     primer_bind     complement(5595..5614)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5596..5614)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        5660..5737
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(5738..6330,1..490)
                     /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(5757..5776)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg
       61 cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt
      121 tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac
      181 cgagtccggg ctgcgcgttg gtgcggacat ctcggtagtg ggatacgacg ataccgaaga
      241 cagctcatgt tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca
      301 aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct
      361 gttgcccgtc tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc
      421 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
      481 cgggcagtga gcgcaacgca attaatgtaa cttagctcac tcattaggca ccgggatctc
      541 gaccgatgcc cttgagagcc ttcaacccag tcagctcctt ccggtgggcg cggggcatga
      601 ctaacatgag aattacaact tatatcgtat ggggctgact tcaggtgcta catttgaaga
      661 gataaattgc actgaaatct agagtaacgg aatagctgtt cgttgacttg atagaccgat
      721 tgattcatca tctcataaat aaagaaaaac caccgctacc aacggtggtt ttctcaaggt
      781 tcgctgagct accaactctt tgaaccaagg taagtgggtt ggaggaccgc actcaccaaa
      841 atctgttctt tcagtttagc cttaacaggt gcataacttc aagacaaagt cctctaaatc
      901 agttaccaat ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
      961 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     1021 gcttggagcg aacgacctac accgaactga gataccaaca gcgtgagcta tgagaaagcg
     1081 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     1141 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     1201 ttcgccacct ctggcttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     1261 ggaaaaacgc ctgcggcgtt ggcttcttcc ggtgctttgc tttttgctca catgttcttt
     1321 ccggctttat cccctgattc tgtggataac cgtattaccg cttttgagtg agctgacacc
     1381 gctcgccgca gtcgaacgac cgagcgtagc gagtcagtga gcgaggaagc ggaagagcgc
     1441 tgcatgctgc ttacgccccg ccctgccact catcgcagta ctgttgtaat tcattaagca
     1501 ttctgccgac atggaagcca tcacaaacgg catgatgaac ctgaatcgcc agcggcatca
     1561 gcaccttgtc gccttgcgta taatatttgc ccatggtgaa aacgggggcg aagaagttgt
     1621 ccatattggc cacgtttaaa tcaaaactgg tgaaactcac ccagggattg gctgagacga
     1681 aaaacatatt ctcaataaac cctttaggga aataggccag gttttcaccg taacacgcca
     1741 catcttgcga atatatgtgt agaaactgcc ggaaatcgtc gtggtattca ctccagagcg
     1801 atgaaaacgt ttcagtttgc tcatggaaaa cggtgtaaca agggtgaaca ctatcccata
     1861 tcaccagctc accgtctttc attgccatac gaaattccgg atgagcattc atcaggcggg
     1921 caagaatgtg aataaaggcc ggataaaact tgtgcttatt tttctttacg gtctttaaaa
     1981 aggccgtaat atccagctga acggtctggt tataggtaca ttgagcaact gactgaaatg
     2041 cctcaaaatg ttctttacga tgccattggg atatatcaac ggtggtatat ccagtgattt
     2101 ttttctccat tttagcttcc ttagctcctg aaaatctcga taactcaaaa aatacgcccg
     2161 gtagtgatct tatttcatta tggtgaaagt tggaacctct tacgtgccga tcacaacgtc
     2221 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     2281 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     2341 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     2401 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     2461 gccgcgctta atgcgccgct acagggcgcg tcccattcgc caatccggat atagttcctc
     2521 ctttcagcaa aaaacccctc aagacccgtt tagaggcccc aaggggttat gctagttatt
     2581 gctcagcggt ggcagcagcc aactcagctt cctttcgggc tttgtttagc agcctaggtt
     2641 agtggtggtg gtggtggtgg tggtgatcca ttgtcgagcc ctgaaaatac aggttttcgg
     2701 taccgtttcc ctgtgccgga cgcagggccg ggcccgcagc gcgcgccggg atgttttcga
     2761 acacttgggt gacgtccggc tgggaaccaa acacaatatc gtagccacga gccagttcgt
     2821 catccgtcag tgcgtgggtg cccgggacga acagttggcg attaaatgcg gccatttcat
     2881 ggtccagcag atccggacct gccagttccc acggtgcata aaccgttgca cgagcacgac
     2941 cgccacgttc ctgcactgct tcgatggtgc gttccgcgcc ttcaatcgtg gtacccggca
     3001 ggccgacgat aacaaaacaa ttcagttcaa taccggattc tttgcaccag gcactcagac
     3061 ggtccagatc catagcggac ttttgttttg cacgcggcag cagtgcgcct tcctgcggat
     3121 ccagcgtttc cagaccgaca ctgatacgaa cgcaacccgc agcgcccata gagcgaatca
     3181 taccctcgct caggtgatga accgtggtcg cacacttcca cggatacggt gaaccacggc
     3241 cggtcagttc gcggcacagt tcttccatcc actttttatc cagcgtgaag gtcggcgcgt
     3301 aaaaagcgac atattcgaac ggtgcggccg caaaagattc ttcgatatac tgaatcgtac
     3361 gttcaacggt cagacggcgt tctttgcgcg tgtaaatcag cggcacttca caatagctgc
     3421 aaccgaccgg acaaccacga gctgccggca cgaccagttc acggcgacgc gggataccct
     3481 agaacttgtt agcgtcatca aagtacagtg cgtcgtagtg accatacgga atttcatccg
     3541 gggtcggcag ataccagtct tctgccggca gcagatcacc gcgacgtttc ggtgctgccc
     3601 acgaaccgtc gcgacgcaga tgaacacccg gtgcatcagc tgcacggccg cgaaaggctt
     3661 cgatggccgc aaccacaccg ctttcgaaat cgccgttttc cacgatggcg tcaatatcaa
     3721 aacgttcgaa aaagcgcgga ttgacaccag acagacggcc aaaggtaaca atcggtgcat
     3781 ccgggttcag tgcacgggcg tagctaacca ggcgacgcag accgtcgatg ccatcaaatt
     3841 cattcataac ggcaatcaca tcataacgtt cctggaacag gcgcgaaccc agatctttcc
     3901 acgtcagacg ttcaactgat gcatcaatgg cggtaacttg cgcacccggg tacacagcac
     3961 gcagatgacc ggtcacctga tacagtgcca ggtgatgacc agcattgaag tatgacagca
     4021 cttgcggcgg ccagatgacc aggattttac gcggttcgtc cacgatttcg cccatatggg
     4081 atccaccaat ctgttctctg tgagcctcaa taatatcgtt atcctccatg tccaaatctt
     4141 caggggtctg atcagcttga attctaatac cgtcgtacaa gaatcttaag gagtccattt
     4201 ccttaccctg tcttttagcg aacgcttcca tcagccttct taaaggagtg gtctttttga
     4261 tcttaaagaa gatttctgaa gatccatcgg acacctttaa attgatgtga gtctcaggct
     4321 tgacttctgg cttgacctct ggcttagctt cttgattgac ttctgagtca ctagtgctcc
     4381 cgaggttgtt gttattgtta ttgttgttgt tgttcgagct cgaattagtc tgcgcgtctt
     4441 tcagggcttc atcgacagtc tgacgaccgc tggcggcgtt gatcaccgca gtacgcacgg
     4501 cataccagaa agcggacatc tgcgggatgt tcggcatgat ttcacctttc tgggcgtttt
     4561 ccatggtggc ggcaatacgt ggatctttcg ccaactcttc ctcgtaagac ttcagcgcta
     4621 cggcacccag cggtttgtct ttattaaccg cttccagacc ttcatcagtc agcagatagt
     4681 tttcgaggaa ctcttttgcc agctctttgt tcggactggc ggcgttaata cctgcgctca
     4741 gcacgccaac gaacggtttg gatggttgac ccttgaaggt cggcagtacc gttacaccat
     4801 aattcacttt gctggtgtcg atgttggacc atgcccacgg gccgttgatg gtcatcgctg
     4861 tttcgccttt attaaaggca gcttctgcga tggagtaatc ggtgtctgca ttcatgtgtt
     4921 tgtttttaat caggtcaacc aggaaggtca gacccgcttt cgcgccagcg ttatccacgc
     4981 ccacgtcttt aatgtcgtac ttgccgtttt catacttgaa cgcataaccc ccgtcagcag
     5041 caatcagcgg ccaggtgaag tacggttctt gcaggttgaa catcagcgcg ctcttacctt
     5101 tcgctttcag ttctttatcc agcgccggga tctcttccca ggtttttggc gggttcggca
     5161 gcagatcttt gttataaatc agcgataacg cttcaacagc gatcgggtaa gcaatcagct
     5221 tgccgttgta acgtacggca tcccaggtaa acggatacag cttgtcctgg aacgctttgt
     5281 ccggggtgat ttcagccaac aggccagatt gagcgtagcc accaaagcgg tcgtgtgccc
     5341 agaagataat gtcagggcca tcgccagttg ccgcaacctg tgggaatttc tcttccagtt
     5401 tatccggatg ctcaacggtg actttaattc cggtatcttt ctcgaatttc ttaccgactt
     5461 cagcgagacc gttatagcct ttatcgccgt taatccagat taccagttta ccttcttcga
     5521 ttttcatggt atatctcctt attaaagtta aacaaaatta tttctacagg ggaattgtta
     5581 tccgctcaca attcccctat agtgagtcgt attaatttcc taatgcagga gtcgcataag
     5641 ggagagcgtc gagatcccgg acaccatcga atggcgcaaa acctttcgcg gtatggcatg
     5701 atagcgcccg gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa cgttatacga
     5761 tgtcgcagag tatgccggtg tctcttatca gaccgtttcc cgcgtggtga accaggccag
     5821 ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc tgaattacat
     5881 tcccaaccgc gtggcacaac aactggcggg caaacagtcg ttgctgattg gcgttgccac
     5941 ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga
     6001 tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa
     6061 agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta actatccgct
     6121 ggatgaccag gatgccattg ctgtggaagc tgcctgcact aatgttccgg cgttatttct
     6181 tgatgtctct gaccagacac ccatcaacag tattattttc tcccatgaag acggtacgcg
     6241 actgggcgtg gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc
     6301 attaagttct gtctcggcgc gtctgcgtct
//
