LOCUS       sequence-342846-        5193 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid with a TEV removable C-term 8xHis Tagged 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 5193)
  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 5193)
  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 SMT3 NikJC199U
FEATURES             Location/Qualifiers
     source          1..5193
                     /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             2816..3106
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             3122..3145
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(3198..3216)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3212..3259
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(3383..3402)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3589..3691
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             3692..4351
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3758..3777)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      complement(4367..5116)
                     /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(4377..4394)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(4617..4636)
                     /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 gcactagtga
     2821 ctcagaagtc aatcaagaag ctaagccaga ggtcaagcca gaagtcaagc ctgagactca
     2881 catcaattta aaggtgtccg atggatcttc agaaatcttc tttaagatca aaaagaccac
     2941 tcctttaaga aggctgatgg aagcgttcgc taaaagacag ggtaaggaaa tggactcctt
     3001 aagattcttg tacgacggta ttagaattca agctgatcag acccctgaag atttggacat
     3061 ggaggataac gatattattg aggctcacag agaacagatt ggtggatcct cgacaatgga
     3121 tcaccaccac caccaccacc accactaacc taggctgcta aacaaagccc gaaaggaagc
     3181 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     3241 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg
     3301 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     3361 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     3421 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     3481 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     3541 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttcttttg atcggcacgt
     3601 aagaggttcc aactttcacc ataatgaaat aagatcacta ccgggcgtat tttttgagtt
     3661 atcgagattt tcaggagcta aggaagctaa aatggagaaa aaaatcactg gatataccac
     3721 cgttgatata tcccaatggc atcgtaaaga acattttgag gcatttcagt cagttgctca
     3781 atgtacctat aaccagaccg ttcagctgga tattacggcc tttttaaaga ccgtaaagaa
     3841 aaataagcac aagttttatc cggcctttat tcacattctt gcccgcctga tgaatgctca
     3901 tccggaattt cgtatggcaa tgaaagacgg tgagctggtg atatgggata gtgttcaccc
     3961 ttgttacacc gttttccatg agcaaactga aacgttttca tcgctctgga gtgaatacca
     4021 cgacgatttc cggcagtttc tacacatata ttcgcaagat gtggcgtgtt acggtgaaaa
     4081 cctggcctat ttccctaaag ggtttattga gaatatgttt ttcgtctcag ccaatccctg
     4141 ggtgagtttc accagttttg atttaaacgt ggccaatatg gacaacttct tcgcccccgt
     4201 tttcaccatg ggcaaatatt atacgcaagg cgacaaggtg ctgatgccgc tggcgattca
     4261 ggttcatcat gccgtttgtg atggcttcca tgtcggcaga atgcttaatg aattacaaca
     4321 gtactgcgat gagtggcagg gcggggcgta agcagcatgc agcgctcttc cgcttcctcg
     4381 ctcactgact cgctacgctc ggtcgttcga ctgcggcgag cggtgtcagc tcactcaaaa
     4441 gcggtaatac ggttatccac agaatcaggg gataaagccg gaaagaacat gtgagcaaaa
     4501 agcaaagcac cggaagaagc caacgccgca ggcgtttttc cataggctcc gcccccctga
     4561 cgagcatcac aaaaatcgac gctcaagcca gaggtggcga aacccgacag gactataaag
     4621 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     4681 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     4741 ctgttggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     4801 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     4861 aagacacgac ttatcgccac tggcagcagc cattggtaac tgatttagag gactttgtct
     4921 tgaagttatg cacctgttaa ggctaaactg aaagaacaga ttttggtgag tgcggtcctc
     4981 caacccactt accttggttc aaagagttgg tagctcagcg aaccttgaga aaaccaccgt
     5041 tggtagcggt ggtttttctt tatttatgag atgatgaatc aatcggtcta tcaagtcaac
     5101 gaacagctat tccgttactc tagatttcag tgcaatttat ctcttcaaat gtagcacctg
     5161 aagtcagccc catacgatat aagttgtaat tct
//
