LOCUS       sequence-203313-        7130 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmids for production of recombinant 
            metallothionein 2a K31A 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_K31A
FEATURES             Location/Qualifiers
     source          1..7130
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2..106
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(107..175,176..967)
                     /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(325..344)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(1009..1522)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1204..1225)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1416..1435
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      1633..2221
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2122..2141
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2375..2392
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2414..2433)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2574..2596
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2590..2781)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3338..4420)
                     /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     4382..4401
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4421..4498)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      4651..4737
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4834..4920
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     5055..5074
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5055..5073
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5074..5098
                     /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             5113..5135
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5188..6006
                     /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             6013..6165
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVP
                     ALWQLQ"
     CDS             6169..6717
                     /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(7011..7029)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7025..7072
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
       61 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
      121 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
      181 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
      241 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgtttccc
      301 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg
      361 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
      421 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
      481 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
      541 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
      601 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
      661 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
      721 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
      781 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
      841 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
      901 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
      961 ttggtaactg tcagaccaag tttactcata tatactttag attgatttac cccggttgat
     1021 aatcagaaaa gccccaaaaa caggaagatt gtataagcaa atatttaaat tgtaaacgtt
     1081 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     1141 gccgaaatcg gcaaaatccc ttataaatca aaagaatagc ccgagatagg gttgagtgtt
     1201 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     1261 aaaaccgtct atcagggcga tggcccacta cgtgaaccat cacccaaatc aagttttttg
     1321 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     1381 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     1441 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     1501 aatgcgccgc tacagggcgc gtaaaaggat ctaggtgaag atcctttttg ataatctcat
     1561 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     1621 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     1681 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     1741 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     1801 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     1861 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     1921 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     1981 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     2041 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     2101 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     2161 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     2221 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     2281 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     2341 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagctat
     2401 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
     2461 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     2521 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     2581 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag
     2641 ctcatcagcg tggtcgtgca gcgattcaca gatgtctgcc tgttcatccg cgtccagctc
     2701 gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc
     2761 ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt
     2821 aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc
     2881 ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag
     2941 aaaaatcact cagggtcaat gccagccgaa cgccagcaag acgtagccca gcgcgtcggc
     3001 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
     3061 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
     3121 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
     3181 tacgagttgc atgataaaga agacagtcat aagtgcagcg acgatagtca tgccccgcgc
     3241 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
     3301 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     3361 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     3421 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
     3481 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
     3541 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
     3601 cgtatcccac taccgagatg tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
     3661 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
     3721 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
     3781 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
     3841 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
     3901 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg
     3961 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
     4021 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
     4081 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca
     4141 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
     4201 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
     4261 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
     4321 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
     4381 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac
     4441 tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg
     4501 ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg
     4561 aggccgttga gcaccgccgc cgcaaggaat ggtgcatgcc ggcatgccgc cctttcgtct
     4621 tcaagaatta attcccaatt ccccaggcat caaataaaac gaaaggctca gtcgaaagac
     4681 tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg
     4741 ccgggagcgg atttgaacgt tgcgaagcaa cggcccggag ggtggcgggc aggacgcccg
     4801 ccataaactg ccaggaatta attccccagg catcaaataa aacgaaaggc tcagtcgaaa
     4861 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat
     4921 ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg ggcaggacgc
     4981 ccgccataaa ctgccaggaa ttggggatcg gaattaattc ccggtttaaa ccggggatct
     5041 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
     5101 tctagaaata attttgttta actttaagaa ggagatatac ttatgaaaat cgaagaaggt
     5161 aaactggtaa tcggttccct ggagggttgc tttgccaagg gtaccaatgt tttaatggcg
     5221 gatgggtcta ttgaatgtat tgaaaacatt gaggttggta ataaggtcat gggtaaagat
     5281 ggcagacctc gtgaggtaat taaattgccc agaggaagag aaactatgta cagcgtcgtg
     5341 cagaaaagtc agcacagagc ccacaaaagt gactcaagtc gtgaagtgcc agaattactc
     5401 aagtttacgt gtaatgcgac ccatgagttg gttgttagaa cacctcgtag tgtccgccgt
     5461 ttgtctcgta ccattaaggg tgtcgaatat tttgaagtta ttacttttga gatgggccaa
     5521 aagaaagccc ccgacggtag aattgttgag cttgtcaagg aagtttcaaa gagctaccca
     5581 atatctgagg ggcctgagag agccaacgaa ttagtagaat cctatagaaa ggcttcaaat
     5641 aaagcttatt ttgagtggac tattgaggcc agagatcttt ctctgttggg ttcccatgtt
     5701 cgtaaagcta cctaccagac ttacgctcca attctttatg agaatgacca ctttttcgac
     5761 tacatgcaaa aaagtaagtt tcatctcacc attgaaggtc caaaagtact tgcttattta
     5821 cttggtttat ggattggtga tggattgtct gacagggcaa ctttttcggt tgattccaga
     5881 gatacttctt tgatggaacg tgttactgaa tatgctgaaa agttgaattt gtgcgccgag
     5941 tataaggaca gaaaagaacc acaagttgcc aaaactgtta atttgtactc taaagttgtc
     6001 agaggtgcta gcacaaatcc tggtgtatcc gcttggcagg tcaacacagc ttatactgcg
     6061 ggacaattgg tcacatataa cggcaagacg tataaatgtt tgcagcccca cacctccttg
     6121 gcaggatggg aaccatccaa cgttcctgcc ttgtggcagc ttcaaggtgg ccacggtggt
     6181 attcgcaata atcttaatac tgagaatcca ttatgggacg ctattgttgg cttaggattc
     6241 ttgaaggacg gtgtcaaaaa tattccttct ttcttgtcta cggacaatat cggtactcgt
     6301 gaaacatttc ttgctggtct aattgattct gatggctatg ttactgatga gcatggtatt
     6361 aaagcaacaa taaagacaat tcatacttct gtcagagatg gtttggtttc ccttgctcgt
     6421 tctttaggct tagtagtctc ggttaacgca gaacctgcta aggttgacat gaatgtcacc
     6481 aaacataaaa ttagttatgc tatttatatg tctggtggag atgttttgct taacgttctt
     6541 tcgaagtgtg ccggctctaa aaaattcagg cctgctcccg ccgctgcttt tgcacgtgag
     6601 tgccgcggat tttatttcga gttacaagaa ttgaaggaag acgattatta tgggattact
     6661 ttatctgatg attctgatca tcagtttttg cttggatctc aggttgttgt acagaacatg
     6721 gatccgaact gcagctgtgc ggcgggtgat tcttgcacgt gtgcgggcag ctgcaaatgc
     6781 aaagaatgca aatgcaccag ctgcaaagca agctgctgca gctgctgtcc ggtgggctgt
     6841 gcgaaatgcg cgcaaggctg catttgcaaa ggcgcgagcg ataaatgcag ctgctgcgcg
     6901 taactgcaga ccaccatcgt cgacggatcc gaattccctg caggtaatta aataactagt
     6961 tgatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
     7021 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
     7081 aggaactata tccggataac tacgtcaggt ggcacttttc ggggaaatgt
//
