LOCUS       sequence-203315-        7313 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmids for production of recombinant 
            metallothionein 2a K20A mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7313)
  AUTHORS   Singh AK, Pomorski A, Wu S, Peris-Diaz MD, Czepczynska-Krezel H, 
            Krezel A
  TITLE     The connection of alpha- and beta-domains in mammalian 
            metallothionein-2 differentiates Zn(II) binding affinities, affects 
            folding, and determines zinc buffering properties.
  JOURNAL   Metallomics. 2023 Jun 1;15(6):mfad029. doi: 10.1093/mtomcs/mfad029.
  PUBMED    37147085
REFERENCE   2  (bases 1 to 7313)
  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: 
            "Metallomics."; date: "2023-06-1"; volume: "15(6):mfad029. doi"; 
            pages: " 10.1093/mtomcs/mfad029"
COMMENT     Sequence Label: MT2a_K20A
FEATURES             Location/Qualifiers
     source          1..7313
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        21..125
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(126..194,195..986)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(344..363)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(1028..1541)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1223..1244)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1435..1454
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      1652..2240
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2141..2160
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2394..2411
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2433..2452)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2593..2615
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2609..2800)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3357..4439)
                     /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     4401..4420
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4440..4517)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      4670..4756
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4853..4939
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5036..5122
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     5257..5276
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5257..5275
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5276..5300
                     /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)."
     RBS             5315..5337
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5390..6208
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae VMA1"
                     /product="5' region of the intein from the yeast Vma1 
                     subunit of the vacuolar ATPase (Chong et al., 1998)"
                     /label=Sce VMA intein 5' region
                     /translation="CFAKGTNVLMADGSIECIENIEVGNKVMGKDGRPREVIKLPRGRE
                     TMYSVVQKSQHRAHKSDSSREVPELLKFTCNATHELVVRTPRSVRRLSRTIKGVEYFEV
                     ITFEMGQKKAPDGRIVELVKEVSKSYPISEGPERANELVESYRKASNKAYFEWTIEARD
                     LSLLGSHVRKATYQTYAPILYENDHFFDYMQKSKFHLTIEGPKVLAYLLGLWIGDGLSD
                     RATFSVDSRDTSLMERVTEYAEKLNLCAEYKDRKEPQVAKTVNLYSKVVRG"
     CDS             6215..6367
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVP
                     ALWQLQ"
     CDS             6371..6919
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae VMA1"
                     /product="modified 3' region of the intein from the yeast 
                     Vma1 subunit of the vacuolar ATPase (Chong et al., 1998)"
                     /label=Sce VMA intein 3' region
                     /note="C-terminal cleavage of this intein can be induced by
                     thiols."
                     /translation="GHGGIRNNLNTENPLWDAIVGLGFLKDGVKNIPSFLSTDNIGTRE
                     TFLAGLIDSDGYVTDEHGIKATIKTIHTSVRDGLVSLARSLGLVVSVNAEPAKVDMNVT
                     KHKISYAIYMSGGDVLLNVLSKCAGSKKFRPAPAAAFARECRGFYFELQELKEDDYYGI
                     TLSDDSDHQFLLGSQVVVQN"
     primer_bind     complement(7213..7231)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7227..7274
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
       61 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
      121 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
      181 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
      241 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
      301 gccccgaaga acgtttccca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
      361 tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
      421 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
      481 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
      541 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
      601 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
      661 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
      721 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
      781 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
      841 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
      901 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
      961 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     1021 ttgatttacc ccggttgata atcagaaaag ccccaaaaac aggaagattg tataagcaaa
     1081 tatttaaatt gtaaacgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     1141 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagcc
     1201 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     1261 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     1321 acccaaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     1381 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     1441 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     1501 caccacaccc gccgcgctta atgcgccgct acagggcgcg taaaaggatc taggtgaaga
     1561 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     1621 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     1681 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     1741 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     1801 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     1861 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     1921 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     1981 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     2041 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     2101 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     2161 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     2221 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     2281 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     2341 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     2401 cagtgagcga ggaagctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     2461 agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc
     2521 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     2581 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     2641 cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgcag cgattcacag atgtctgcct
     2701 gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa
     2761 agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg
     2821 gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg
     2881 ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat
     2941 ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagccgaac gccagcaaga
     3001 cgtagcccag cgcgtcggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt
     3061 tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa
     3121 gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga
     3181 gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga
     3241 cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     3301 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     3361 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     3421 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     3481 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     3541 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     3601 gagctgtctt cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg
     3661 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     3721 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     3781 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     3841 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     3901 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     3961 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     4021 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     4081 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     4141 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     4201 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     4261 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     4321 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     4381 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     4441 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     4501 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     4561 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgccg
     4621 gcatgccgcc ctttcgtctt caagaattaa ttcccaattc cccaggcatc aaataaaacg
     4681 aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct
     4741 cctgagtagg acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg
     4801 gtggcgggca ggacgcccgc cataaactgc caggaattaa ttccccaggc atcaaataaa
     4861 acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc
     4921 tctcctgagt aggacaaatc cgccgggagc ggatttgaac gttgcgaagc aacggcccgg
     4981 agggtggcgg gcaggacgcc cgccataaac tgccaggaat taattcccca ggcatcaaat
     5041 aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa
     5101 cgctctcctg agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc
     5161 cggagggtgg cgggcaggac gcccgccata aactgccagg aattggggat cggaattaat
     5221 tcccggttta aaccggggat ctcgatcccg cgaaattaat acgactcact ataggggaat
     5281 tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat
     5341 acttatgaaa atcgaagaag gtaaactggt aatcggttcc ctggagggtt gctttgccaa
     5401 gggtaccaat gttttaatgg cggatgggtc tattgaatgt attgaaaaca ttgaggttgg
     5461 taataaggtc atgggtaaag atggcagacc tcgtgaggta attaaattgc ccagaggaag
     5521 agaaactatg tacagcgtcg tgcagaaaag tcagcacaga gcccacaaaa gtgactcaag
     5581 tcgtgaagtg ccagaattac tcaagtttac gtgtaatgcg acccatgagt tggttgttag
     5641 aacacctcgt agtgtccgcc gtttgtctcg taccattaag ggtgtcgaat attttgaagt
     5701 tattactttt gagatgggcc aaaagaaagc ccccgacggt agaattgttg agcttgtcaa
     5761 ggaagtttca aagagctacc caatatctga ggggcctgag agagccaacg aattagtaga
     5821 atcctataga aaggcttcaa ataaagctta ttttgagtgg actattgagg ccagagatct
     5881 ttctctgttg ggttcccatg ttcgtaaagc tacctaccag acttacgctc caattcttta
     5941 tgagaatgac cactttttcg actacatgca aaaaagtaag tttcatctca ccattgaagg
     6001 tccaaaagta cttgcttatt tacttggttt atggattggt gatggattgt ctgacagggc
     6061 aactttttcg gttgattcca gagatacttc tttgatggaa cgtgttactg aatatgctga
     6121 aaagttgaat ttgtgcgccg agtataagga cagaaaagaa ccacaagttg ccaaaactgt
     6181 taatttgtac tctaaagttg tcagaggtgc tagcacaaat cctggtgtat ccgcttggca
     6241 ggtcaacaca gcttatactg cgggacaatt ggtcacatat aacggcaaga cgtataaatg
     6301 tttgcagccc cacacctcct tggcaggatg ggaaccatcc aacgttcctg ccttgtggca
     6361 gcttcaaggt ggccacggtg gtattcgcaa taatcttaat actgagaatc cattatggga
     6421 cgctattgtt ggcttaggat tcttgaagga cggtgtcaaa aatattcctt ctttcttgtc
     6481 tacggacaat atcggtactc gtgaaacatt tcttgctggt ctaattgatt ctgatggcta
     6541 tgttactgat gagcatggta ttaaagcaac aataaagaca attcatactt ctgtcagaga
     6601 tggtttggtt tcccttgctc gttctttagg cttagtagtc tcggttaacg cagaacctgc
     6661 taaggttgac atgaatgtca ccaaacataa aattagttat gctatttata tgtctggtgg
     6721 agatgttttg cttaacgttc tttcgaagtg tgccggctct aaaaaattca ggcctgctcc
     6781 cgccgctgct tttgcacgtg agtgccgcgg attttatttc gagttacaag aattgaagga
     6841 agacgattat tatgggatta ctttatctga tgattctgat catcagtttt tgcttggatc
     6901 tcaggttgtt gtacagaaca tggatccgaa ctgcagctgt gcggcgggtg attcttgcac
     6961 gtgtgcgggc agctgcgcgt gcaaagaatg caaatgcacc agctgcaaaa aaagctgctg
     7021 cagctgctgt ccggtgggct gtgcgaaatg cgcgcaaggc tgcatttgca aaggcgcgag
     7081 cgataaatgc agctgctgcg cgtaactgca gaccaccatc gtcgacggat ccgaattccc
     7141 tgcaggtaat taaataacta gttgatccgg ctgctaacaa agcccgaaag gaagctgagt
     7201 tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
     7261 tgaggggttt tttgctgaaa ggaggaacta tatccggata actacgtcag gtg
//
