LOCUS       sequence-394896-        4113 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmids for sortase mediated purification of 
            recombinant MT3. Contains His-tag for purification and SUMO-tag for 
            better solubility and LPETG for sortase mediated cleavage.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4113)
  AUTHORS   Singh AK, Krezel A
  TITLE     Calcium-assisted sortase A cleavage of SUMOylated metallothionein 
            constructs leads to high-yield production of human MT3.
  JOURNAL   Microb Cell Fact. 2023 Jul 11;22(1):125. doi: 
            10.1186/s12934-023-02134-x.
  PUBMED    37434134
REFERENCE   2  (bases 1 to 4113)
  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: "Microb Cell
            Fact."; date: "2023-07-11"; volume: "22(1):125. doi"; pages: " 
            10.1186/s12934-023-02134-x"
COMMENT     Sequence Label: SUMO_LPETG_MT3
FEATURES             Location/Qualifiers
     source          1..4113
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             124..414
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             667..711
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(749..767)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      763..810
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1040..1855)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1040..1855)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1763..1782
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1856..1947)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1963..2712)
                     /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(1973..1990)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2213..2232)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2908..3990)
                     /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     3952..3971
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3991..4068)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctgta gaaataattt
       61 tgtttaactt taataaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 actagtgact cagaagtcaa tcaagaagct aagccagagg tcaagccaga agtcaagcct
      181 gagactcaca tcaatttaaa ggtgtccgat ggatcttcag aaatcttctt taagatcaaa
      241 aagaccactc ctttaagaag gctgatggaa gcgttcgcta aaagacaggg taaggaaatg
      301 gactccttaa gattcttgta cgacggtatt agaattcaag ctgatcagac ccctgaagat
      361 ttggacatgg aggataacga tattattgag gctcacagag aacagattgg tggatccctg
      421 ccggaaaccg gcatggaccc ggaaacctgc ccgtgcccgt ctggtggctc ctgtacctgt
      481 gccgattcgt gtaaatgtga aggctgtaaa tgtacctcat gcaaaaagag ctgctgttct
      541 tgctgtccgg cggaatgcga aaaatgtgca aaggattgcg tgtgtaaagg cggtgaagcg
      601 gccgaagcag aagctgaaaa gtgtagttgc tgtcagtaaa agcttgcggc cgcactcgag
      661 tctggtaaag aaaccgctgc tgcgaaattt gaacgccagc acatggactc gtctactagc
      721 gcagcttaat taacctaggc tgctgccacc gctgagcaat aactagcata accccttggg
      781 gcctctaaac gggtcttgag gggttttttg ctgaaacctc aggcatttga gaagcacacg
      841 gtcacactgc ttccggtagt caataaaccg gtaaaccagc aatagacata agcggctatt
      901 taacgaccct gccctgaacc gacgacaagc tgacgaccgg gtctccgcaa gtggcacttt
      961 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     1021 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
     1081 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     1141 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     1201 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     1261 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
     1321 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
     1381 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcggtcgctg ttaaaaggac
     1441 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     1501 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     1561 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     1621 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     1681 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     1741 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     1801 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcatactct
     1861 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     1921 ttgaatgtat ttagaaaaat aaacaaatag gcatgcagcg ctcttccgct tcctcgctca
     1981 ctgactcgct acgctcggtc gttcgactgc ggcgagcggt gtcagctcac tcaaaagcgg
     2041 taatacggtt atccacagaa tcaggggata aagccggaaa gaacatgtga gcaaaaagca
     2101 aagcaccgga agaagccaac gccgcaggcg tttttccata ggctccgccc ccctgacgag
     2161 catcacaaaa atcgacgctc aagccagagg tggcgaaacc cgacaggact ataaagatac
     2221 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     2281 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     2341 tggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     2401 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     2461 cacgacttat cgccactggc agcagccatt ggtaactgat ttagaggact ttgtcttgaa
     2521 gttatgcacc tgttaaggct aaactgaaag aacagatttt ggtgagtgcg gtcctccaac
     2581 ccacttacct tggttcaaag agttggtagc tcagcgaacc ttgagaaaac caccgttggt
     2641 agcggtggtt tttctttatt tatgagatga tgaatcaatc ggtctatcaa gtcaacgaac
     2701 agctattccg ttactctaga tttcagtgca atttatctct tcaaatgtag cacctgaagt
     2761 cagccccata cgatataagt tgtaattctc atgttagtca tgccccgcgc ccaccggaag
     2821 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
     2881 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     2941 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     3001 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
     3061 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     3121 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
     3181 taccgagatg tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     3241 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     3301 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     3361 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     3421 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     3481 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
     3541 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     3601 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     3661 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     3721 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     3781 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     3841 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     3901 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     3961 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     4021 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac
     4081 gctctccctt atgcgactcc tgcattagga aat
//
