LOCUS       sequence-342456-        5184 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid with a TEV removable C-term 8xHis Tag, an N-term 
            SUMO 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 5184)
  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 5184)
  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 SMT3 NikJC199U
FEATURES             Location/Qualifiers
     source          1..5184
                     /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             1421..1711
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             3095..3115
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3128..3151
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(3204..3222)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3218..3265
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(3389..3408)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3580..3682
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             3683..4342
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3749..3768)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      complement(4358..5107)
                     /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(4368..4385)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(4608..4627)
                     /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 catgagcact agtgactcag aagtcaatca
     1441 agaagctaag ccagaggtca agccagaagt caagcctgag actcacatca atttaaaggt
     1501 gtccgatgga tcttcagaaa tcttctttaa gatcaaaaag accactcctt taagaaggct
     1561 gatggaagcg ttcgctaaaa gacagggtaa ggaaatggac tccttaagat tcttgtacga
     1621 cggtattaga attcaagctg atcagacccc tgaagatttg gacatggagg ataacgatat
     1681 tattgaggct cacagagaac agattggtgg atcccatatg ggcgaaatcg tggacgaacc
     1741 gcgtaaaatc ctggtcatct ggccgccgca agtgctgtca tacttcaatg ctggtcatca
     1801 cctggcactg tatcaggtga ccggtcatct gcgtgctgtg tacccgggtg cgcaagttac
     1861 cgccattgat gcatcagttg aacgtctgac gtggaaagat ctgggttcgc gcctgttcca
     1921 ggaacgttat gatgtgattg ccgttatgaa tgaatttgat ggcatcgacg gtctgcgtcg
     1981 cctggttagc tacgcccgtg cactgaaccc ggatgcaccg attgttacct ttggccgtct
     2041 gtctggtgtc aatccgcgct ttttcgaacg ttttgatatt gacgccatcg tggaaaacgg
     2101 cgatttcgaa agcggtgtgg ttgcggccat cgaagccttt cgcggccgtg cagctgatgc
     2161 accgggtgtt catctgcgtc gcgacggttc gtgggcagca ccgaaacgtc gcggtgatct
     2221 gctgccggca gaagactggt atctgccgac cccggatgaa attccgtatg gtcactacga
     2281 cgcactgtac tttgatgacg ctaacaagtt ctagggtatc ccgcgtcgcc gtgaactggt
     2341 cgtgccggca gctcgtggtt gtccggtcgg ttgcagctat tgtgaagtgc cgctgattta
     2401 cacgcgcaaa gaacgccgtc tgaccgttga acgtacgatt cagtatatcg aagaatcttt
     2461 tgcggccgca ccgttcgaat atgtcgcttt ttacgcgccg accttcacgc tggataaaaa
     2521 gtggatggaa gaactgtgcc gcgaactgac cggccgtggt tcaccgtatc cgtggaagtg
     2581 tgcgaccacg gttcatcacc tgagcgaggg tatgattcgc tctatgggcg ctgcgggttg
     2641 cgttcgtatc agtgtcggtc tggaaacgct ggatccgcag gaaggcgcac tgctgccgcg
     2701 tgcaaaacaa aagtccgcta tggatctgga ccgtctgagt gcctggtgca aagaatccgg
     2761 tattgaactg aattgttttg ttatcgtcgg cctgccgggt accacgattg aaggcgcgga
     2821 acgcaccatc gaagcagtgc aggaacgtgg cggtcgtgct cgtgcaacgg tttatgcacc
     2881 gtgggaactg gcaggtccgg atctgctgga ccatgaaatg gccgcattta atcgccaact
     2941 gttcgtcccg ggcacccacg cactgacgga tgacgaactg gctcgtggct acgatattgt
     3001 gtttggttcc cagccggacg tcacccaagt gttcgaaaac atcccggcgc gcgctgcggg
     3061 cccggccctg cgtccggcac agggaaacgg taccgaaaac ctgtattttc agggctcgac
     3121 aatggatcac caccaccacc accaccacca ctaacctagg ctgctaaaca aagcccgaaa
     3181 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     3241 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg
     3301 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     3361 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     3421 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     3481 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     3541 ccatcgccct gatagacggt ttttcgccct ttgacgttgt gatcggcacg taagaggttc
     3601 caactttcac cataatgaaa taagatcact accgggcgta ttttttgagt tatcgagatt
     3661 ttcaggagct aaggaagcta aaatggagaa aaaaatcact ggatatacca ccgttgatat
     3721 atcccaatgg catcgtaaag aacattttga ggcatttcag tcagttgctc aatgtaccta
     3781 taaccagacc gttcagctgg atattacggc ctttttaaag accgtaaaga aaaataagca
     3841 caagttttat ccggccttta ttcacattct tgcccgcctg atgaatgctc atccggaatt
     3901 tcgtatggca atgaaagacg gtgagctggt gatatgggat agtgttcacc cttgttacac
     3961 cgttttccat gagcaaactg aaacgttttc atcgctctgg agtgaatacc acgacgattt
     4021 ccggcagttt ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa acctggccta
     4081 tttccctaaa gggtttattg agaatatgtt tttcgtctca gccaatccct gggtgagttt
     4141 caccagtttt gatttaaacg tggccaatat ggacaacttc ttcgcccccg ttttcaccat
     4201 gggcaaatat tatacgcaag gcgacaaggt gctgatgccg ctggcgattc aggttcatca
     4261 tgccgtttgt gatggcttcc atgtcggcag aatgcttaat gaattacaac agtactgcga
     4321 tgagtggcag ggcggggcgt aagcagcatg cagcgctctt ccgcttcctc gctcactgac
     4381 tcgctacgct cggtcgttcg actgcggcga gcggtgtcag ctcactcaaa agcggtaata
     4441 cggttatcca cagaatcagg ggataaagcc ggaaagaaca tgtgagcaaa aagcaaagca
     4501 ccggaagaag ccaacgccgc aggcgttttt ccataggctc cgcccccctg acgagcatca
     4561 caaaaatcga cgctcaagcc agaggtggcg aaacccgaca ggactataaa gataccaggc
     4621 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     4681 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgttggta
     4741 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     4801 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     4861 cttatcgcca ctggcagcag ccattggtaa ctgatttaga ggactttgtc ttgaagttat
     4921 gcacctgtta aggctaaact gaaagaacag attttggtga gtgcggtcct ccaacccact
     4981 taccttggtt caaagagttg gtagctcagc gaaccttgag aaaaccaccg ttggtagcgg
     5041 tggtttttct ttatttatga gatgatgaat caatcggtct atcaagtcaa cgaacagcta
     5101 ttccgttact ctagatttca gtgcaattta tctcttcaaa tgtagcacct gaagtcagcc
     5161 ccatacgata taagttgtaa ttct
//
