LOCUS       sequence-394864-        4098 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmids for production of recombinant 
            metallothionein 3 containing N-terminal tags, which can be cleaved 
            later: His-tag for purification and SUMO-tag for better solubility.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4098)
  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 4098)
  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_MT3
FEATURES             Location/Qualifiers
     source          1..4098
                     /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             652..696
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(734..752)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      748..795
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1025..1840)
                     /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(1025..1840)
                     /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     1748..1767
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1841..1932)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1948..2697)
                     /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(1958..1975)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2198..2217)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2893..3975)
                     /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     3937..3956
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3976..4053)
                     /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 tggatccatg
      421 gacccggaaa cctgcccgtg cccgtctggt ggctcctgta cctgtgccga ttcgtgtaaa
      481 tgtgaaggct gtaaatgtac ctcatgcaaa aagagctgct gttcttgctg tccggcggaa
      541 tgcgaaaaat gtgcaaagga ttgcgtgtgt aaaggcggtg aagcggccga agcagaagct
      601 gaaaagtgta gttgctgtca gtaaaagctt gcggccgcac tcgagtctgg taaagaaacc
      661 gctgctgcga aatttgaacg ccagcacatg gactcgtcta ctagcgcagc ttaattaacc
      721 taggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
      781 ttgaggggtt ttttgctgaa acctcaggca tttgagaagc acacggtcac actgcttccg
      841 gtagtcaata aaccggtaaa ccagcaatag acataagcgg ctatttaacg accctgccct
      901 gaaccgacga caagctgacg accgggtctc cgcaagtggc acttttcggg gaaatgtgcg
      961 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta
     1021 attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat
     1081 caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt
     1141 tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac
     1201 aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga
     1261 cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag
     1321 gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg
     1381 attgcgcctg agcgagacga aatacgcggt cgctgttaaa aggacaatta caaacaggaa
     1441 tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag
     1501 gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg
     1561 catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc
     1621 agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca
     1681 gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc
     1741 cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc
     1801 gcggcctaga gcaagacgtt tcccgttgaa tatggctcat actcttcctt tttcaatatt
     1861 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     1921 aaaataaaca aataggcatg cagcgctctt ccgcttcctc gctcactgac tcgctacgct
     1981 cggtcgttcg actgcggcga gcggtgtcag ctcactcaaa agcggtaata cggttatcca
     2041 cagaatcagg ggataaagcc ggaaagaaca tgtgagcaaa aagcaaagca ccggaagaag
     2101 ccaacgccgc aggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     2161 cgctcaagcc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     2221 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     2281 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgttggta tctcagttcg
     2341 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     2401 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     2461 ctggcagcag ccattggtaa ctgatttaga ggactttgtc ttgaagttat gcacctgtta
     2521 aggctaaact gaaagaacag attttggtga gtgcggtcct ccaacccact taccttggtt
     2581 caaagagttg gtagctcagc gaaccttgag aaaaccaccg ttggtagcgg tggtttttct
     2641 ttatttatga gatgatgaat caatcggtct atcaagtcaa cgaacagcta ttccgttact
     2701 ctagatttca gtgcaattta tctcttcaaa tgtagcacct gaagtcagcc ccatacgata
     2761 taagttgtaa ttctcatgtt agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     2821 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     2881 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     2941 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     3001 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     3061 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     3121 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatgtccgc
     3181 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     3241 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     3301 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     3361 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     3421 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     3481 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     3541 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     3601 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     3661 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     3721 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     3781 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     3841 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     3901 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     3961 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     4021 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     4081 actcctgcat taggaaat
//
