LOCUS       sequence-203312-        7130 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmids for production of recombinant 
            metallothionein 2a K30A mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7130)
  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 7130)
  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_K30A
FEATURES             Location/Qualifiers
     source          1..7130
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(93..161,162..953)
                     /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(311..330)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(995..1508)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1190..1211)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1402..1421
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      1619..2207
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2108..2127
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2361..2378
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2400..2419)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2560..2582
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2576..2767)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3324..4406)
                     /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     4368..4387
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4407..4484)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      4637..4723
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4820..4906
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     5041..5060
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5041..5059
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5060..5084
                     /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             5099..5121
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5174..5992
                     /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             5999..6151
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVP
                     ALWQLQ"
     CDS             6155..6703
                     /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(6997..7015)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7011..7058
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        join(7118..7130,1..92)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
       61 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
      121 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
      181 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
      241 acagcggtaa gatccttgag agttttcgcc ccgaagaacg tttcccaatg atgagcactt
      301 ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg
      361 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
      421 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
      481 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
      541 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
      601 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
      661 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
      721 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
      781 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
      841 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
      901 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
      961 accaagttta ctcatatata ctttagattg atttaccccg gttgataatc agaaaagccc
     1021 caaaaacagg aagattgtat aagcaaatat ttaaattgta aacgttaata ttttgttaaa
     1081 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
     1141 aatcccttat aaatcaaaag aatagcccga gatagggttg agtgttgttc cagtttggaa
     1201 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     1261 gggcgatggc ccactacgtg aaccatcacc caaatcaagt tttttggggt cgaggtgccg
     1321 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     1381 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
     1441 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     1501 gggcgcgtaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     1561 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     1621 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     1681 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     1741 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     1801 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     1861 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     1921 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     1981 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     2041 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     2101 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     2161 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     2221 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     2281 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     2341 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agctatggtg cactctcagt
     2401 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     2461 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     2521 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     2581 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     2641 cgtgcagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     2701 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     2761 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     2821 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     2881 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     2941 gtcaatgcca gccgaacgcc agcaagacgt agcccagcgc gtcggccgcc atgccggcga
     3001 taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga
     3061 gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa
     3121 agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga
     3181 taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc
     3241 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     3301 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     3361 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     3421 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
     3481 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     3541 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     3601 gagatgtccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     3661 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     3721 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     3781 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     3841 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     3901 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     3961 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     4021 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     4081 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     4141 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     4201 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     4261 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     4321 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     4381 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     4441 tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc
     4501 tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac
     4561 cgccgccgca aggaatggtg catgccggca tgccgccctt tcgtcttcaa gaattaattc
     4621 ccaattcccc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt
     4681 tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt
     4741 gaacgttgcg aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag
     4801 gaattaattc cccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg
     4861 ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cgggagcgga
     4921 tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc cataaactgc
     4981 caggaattgg ggatcggaat taattcccgg tttaaaccgg ggatctcgat cccgcgaaat
     5041 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
     5101 tgtttaactt taagaaggag atatacttat gaaaatcgaa gaaggtaaac tggtaatcgg
     5161 ttccctggag ggttgctttg ccaagggtac caatgtttta atggcggatg ggtctattga
     5221 atgtattgaa aacattgagg ttggtaataa ggtcatgggt aaagatggca gacctcgtga
     5281 ggtaattaaa ttgcccagag gaagagaaac tatgtacagc gtcgtgcaga aaagtcagca
     5341 cagagcccac aaaagtgact caagtcgtga agtgccagaa ttactcaagt ttacgtgtaa
     5401 tgcgacccat gagttggttg ttagaacacc tcgtagtgtc cgccgtttgt ctcgtaccat
     5461 taagggtgtc gaatattttg aagttattac ttttgagatg ggccaaaaga aagcccccga
     5521 cggtagaatt gttgagcttg tcaaggaagt ttcaaagagc tacccaatat ctgaggggcc
     5581 tgagagagcc aacgaattag tagaatccta tagaaaggct tcaaataaag cttattttga
     5641 gtggactatt gaggccagag atctttctct gttgggttcc catgttcgta aagctaccta
     5701 ccagacttac gctccaattc tttatgagaa tgaccacttt ttcgactaca tgcaaaaaag
     5761 taagtttcat ctcaccattg aaggtccaaa agtacttgct tatttacttg gtttatggat
     5821 tggtgatgga ttgtctgaca gggcaacttt ttcggttgat tccagagata cttctttgat
     5881 ggaacgtgtt actgaatatg ctgaaaagtt gaatttgtgc gccgagtata aggacagaaa
     5941 agaaccacaa gttgccaaaa ctgttaattt gtactctaaa gttgtcagag gtgctagcac
     6001 aaatcctggt gtatccgctt ggcaggtcaa cacagcttat actgcgggac aattggtcac
     6061 atataacggc aagacgtata aatgtttgca gccccacacc tccttggcag gatgggaacc
     6121 atccaacgtt cctgccttgt ggcagcttca aggtggccac ggtggtattc gcaataatct
     6181 taatactgag aatccattat gggacgctat tgttggctta ggattcttga aggacggtgt
     6241 caaaaatatt ccttctttct tgtctacgga caatatcggt actcgtgaaa catttcttgc
     6301 tggtctaatt gattctgatg gctatgttac tgatgagcat ggtattaaag caacaataaa
     6361 gacaattcat acttctgtca gagatggttt ggtttccctt gctcgttctt taggcttagt
     6421 agtctcggtt aacgcagaac ctgctaaggt tgacatgaat gtcaccaaac ataaaattag
     6481 ttatgctatt tatatgtctg gtggagatgt tttgcttaac gttctttcga agtgtgccgg
     6541 ctctaaaaaa ttcaggcctg ctcccgccgc tgcttttgca cgtgagtgcc gcggatttta
     6601 tttcgagtta caagaattga aggaagacga ttattatggg attactttat ctgatgattc
     6661 tgatcatcag tttttgcttg gatctcaggt tgttgtacag aacatggatc cgaactgcag
     6721 ctgtgcggcg ggtgattctt gcacgtgtgc gggcagctgc aaatgcaaag aatgcaaatg
     6781 caccagctgc gcaaaaagct gctgcagctg ctgtccggtg ggctgtgcga aatgcgcgca
     6841 aggctgcatt tgcaaaggcg cgagcgataa atgcagctgc tgcgcgtaac tgcagaccac
     6901 catcgtcgac ggatccgaat tccctgcagg taattaaata actagttgat ccggctgcta
     6961 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
     7021 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     7081 gataactacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
//
