LOCUS       sequence-203317-        7130 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmids for production of recombinant 
            metallothionein 2a S6A 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_S6A
FEATURES             Location/Qualifiers
     source          1..7130
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        7..111
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(112..180,181..972)
                     /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(330..349)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(1014..1527)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1209..1230)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1421..1440
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      1638..2226
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2127..2146
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2380..2397
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2419..2438)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2579..2601
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2595..2786)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3343..4425)
                     /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     4387..4406
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4426..4503)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      4656..4742
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4839..4925
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     5060..5079
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5060..5078
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5079..5103
                     /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             5118..5140
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5193..6011
                     /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             6018..6170
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVP
                     ALWQLQ"
     CDS             6174..6722
                     /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(7016..7034)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7030..7077
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
       61 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
      121 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
      181 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
      241 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
      301 ttcccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac
      361 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
      421 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
      481 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
      541 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
      601 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
      661 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
      721 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
      781 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
      841 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
      901 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
      961 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaccccgg
     1021 ttgataatca gaaaagcccc aaaaacagga agattgtata agcaaatatt taaattgtaa
     1081 acgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     1141 aataggccga aatcggcaaa atcccttata aatcaaaaga atagcccgag atagggttga
     1201 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     1261 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc aaatcaagtt
     1321 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     1381 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     1441 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     1501 cgcttaatgc gccgctacag ggcgcgtaaa aggatctagg tgaagatcct ttttgataat
     1561 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     1621 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     1681 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     1741 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     1801 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     1861 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     1921 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     1981 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     2041 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     2101 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     2161 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     2221 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     2281 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     2341 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     2401 gctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
     2461 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
     2521 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     2581 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg
     2641 taaagctcat cagcgtggtc gtgcagcgat tcacagatgt ctgcctgttc atccgcgtcc
     2701 agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta
     2761 agggcggttt tttcctgttt ggtcactgat gcctccgcgt aagggggatt tctgttcatg
     2821 ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa
     2881 catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac
     2941 cagagaaaaa tcactcaggg tcaatgccag ccgaacgcca gcaagacgta gcccagcgcg
     3001 tcggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca
     3061 gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc
     3121 atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc
     3181 tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc
     3241 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc
     3301 ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag
     3361 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3421 ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt
     3481 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag
     3541 caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt
     3601 atcgtcgtat cccactaccg agatgtccgc accaacgcgc agcccggact cggtaatggc
     3661 gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc
     3721 ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg
     3781 ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag
     3841 acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc
     3901 gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat
     3961 gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac
     4021 agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc
     4081 gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac
     4141 caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg
     4201 cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag
     4261 ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc
     4321 ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga
     4381 gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa
     4441 ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt
     4501 gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta
     4561 ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgccggcat gccgcccttt
     4621 cgtcttcaag aattaattcc caattcccca ggcatcaaat aaaacgaaag gctcagtcga
     4681 aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa
     4741 atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg cgggcaggac
     4801 gcccgccata aactgccagg aattaattcc ccaggcatca aataaaacga aaggctcagt
     4861 cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga
     4921 caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag
     4981 gacgcccgcc ataaactgcc aggaattggg gatcggaatt aattcccggt ttaaaccggg
     5041 gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat
     5101 tcccctctag aaataatttt gtttaacttt aagaaggaga tatacttatg aaaatcgaag
     5161 aaggtaaact ggtaatcggt tccctggagg gttgctttgc caagggtacc aatgttttaa
     5221 tggcggatgg gtctattgaa tgtattgaaa acattgaggt tggtaataag gtcatgggta
     5281 aagatggcag acctcgtgag gtaattaaat tgcccagagg aagagaaact atgtacagcg
     5341 tcgtgcagaa aagtcagcac agagcccaca aaagtgactc aagtcgtgaa gtgccagaat
     5401 tactcaagtt tacgtgtaat gcgacccatg agttggttgt tagaacacct cgtagtgtcc
     5461 gccgtttgtc tcgtaccatt aagggtgtcg aatattttga agttattact tttgagatgg
     5521 gccaaaagaa agcccccgac ggtagaattg ttgagcttgt caaggaagtt tcaaagagct
     5581 acccaatatc tgaggggcct gagagagcca acgaattagt agaatcctat agaaaggctt
     5641 caaataaagc ttattttgag tggactattg aggccagaga tctttctctg ttgggttccc
     5701 atgttcgtaa agctacctac cagacttacg ctccaattct ttatgagaat gaccactttt
     5761 tcgactacat gcaaaaaagt aagtttcatc tcaccattga aggtccaaaa gtacttgctt
     5821 atttacttgg tttatggatt ggtgatggat tgtctgacag ggcaactttt tcggttgatt
     5881 ccagagatac ttctttgatg gaacgtgtta ctgaatatgc tgaaaagttg aatttgtgcg
     5941 ccgagtataa ggacagaaaa gaaccacaag ttgccaaaac tgttaatttg tactctaaag
     6001 ttgtcagagg tgctagcaca aatcctggtg tatccgcttg gcaggtcaac acagcttata
     6061 ctgcgggaca attggtcaca tataacggca agacgtataa atgtttgcag ccccacacct
     6121 ccttggcagg atgggaacca tccaacgttc ctgccttgtg gcagcttcaa ggtggccacg
     6181 gtggtattcg caataatctt aatactgaga atccattatg ggacgctatt gttggcttag
     6241 gattcttgaa ggacggtgtc aaaaatattc cttctttctt gtctacggac aatatcggta
     6301 ctcgtgaaac atttcttgct ggtctaattg attctgatgg ctatgttact gatgagcatg
     6361 gtattaaagc aacaataaag acaattcata cttctgtcag agatggtttg gtttcccttg
     6421 ctcgttcttt aggcttagta gtctcggtta acgcagaacc tgctaaggtt gacatgaatg
     6481 tcaccaaaca taaaattagt tatgctattt atatgtctgg tggagatgtt ttgcttaacg
     6541 ttctttcgaa gtgtgccggc tctaaaaaat tcaggcctgc tcccgccgct gcttttgcac
     6601 gtgagtgccg cggattttat ttcgagttac aagaattgaa ggaagacgat tattatggga
     6661 ttactttatc tgatgattct gatcatcagt ttttgcttgg atctcaggtt gttgtacaga
     6721 acatggatcc gaactgcgcc tgtgcggcgg gtgattcttg cacgtgtgcg ggcagctgca
     6781 aatgcaaaga atgcaaatgc accagctgca aaaaaagctg ctgcagctgc tgtccggtgg
     6841 gctgtgcgaa atgcgcgcaa ggctgcattt gcaaaggcgc gagcgataaa tgcagctgct
     6901 gcgcgtaact gcagaccacc atcgtcgacg gatccgaatt ccctgcaggt aattaaataa
     6961 ctagttgatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     7021 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     7081 aaaggaggaa ctatatccgg ataactacgt caggtggcac ttttcgggga
//
