LOCUS       sequence-394918-        7688 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmids for production of recombinant fusion 
            protein having beta-domain of metallothionein 2a and chicken Villin 
            headpiece domain HP-35.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7688)
  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 7688)
  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_beta-Villin
FEATURES             Location/Qualifiers
     source          1..7688
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        13..117
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(118..186,187..978)
                     /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(336..355)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(1020..1533)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1215..1236)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1427..1446
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      1644..2232
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2133..2152
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2386..2403
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2425..2444)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2585..2607
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2601..2792)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3349..4431)
                     /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     4393..4412
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4432..4509)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      4662..4748
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4845..4931
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5028..5114
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5211..5297
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5394..5480
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     5615..5634
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5615..5633
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5634..5658
                     /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             5673..5695
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5748..6566
                     /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             6573..6725
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVP
                     ALWQLQ"
     CDS             6729..7277
                     /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(7580..7598)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7594..7641
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
       61 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
      121 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
      181 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
      241 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
      301 gaacgtttcc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
      361 gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
      421 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
      481 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
      541 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
      601 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
      661 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
      721 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
      781 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
      841 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
      901 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
      961 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     1021 ccccggttga taatcagaaa agccccaaaa acaggaagat tgtataagca aatatttaaa
     1081 ttgtaaacgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     1141 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag cccgagatag
     1201 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     1261 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcacccaaat
     1321 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     1381 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     1441 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     1501 ccgccgcgct taatgcgccg ctacagggcg cgtaaaagga tctaggtgaa gatccttttt
     1561 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     1621 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     1681 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     1741 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     1801 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     1861 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     1921 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     1981 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     2041 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     2101 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     2161 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     2221 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     2281 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     2341 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     2401 gaggaagcta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     2461 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc
     2521 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     2581 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
     2641 ctgcggtaaa gctcatcagc gtggtcgtgc agcgattcac agatgtctgc ctgttcatcc
     2701 gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc
     2761 atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg
     2821 ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat
     2881 gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg
     2941 cgggaccaga gaaaaatcac tcagggtcaa tgccagccga acgccagcaa gacgtagccc
     3001 agcgcgtcgg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg
     3061 ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg
     3121 ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc
     3181 ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc
     3241 atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga
     3301 gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     3361 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     3421 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     3481 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     3541 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     3601 ttcggtatcg tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt
     3661 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     3721 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     3781 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     3841 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     3901 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     3961 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     4021 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     4081 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     4141 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     4201 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     4261 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     4321 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     4381 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     4441 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     4501 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
     4561 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc cggcatgccg
     4621 ccctttcgtc ttcaagaatt aattcccaat tccccaggca tcaaataaaa cgaaaggctc
     4681 agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta
     4741 ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca acggcccgga gggtggcggg
     4801 caggacgccc gccataaact gccaggaatt aattccccag gcatcaaata aaacgaaagg
     4861 ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga
     4921 gtaggacaaa tccgccggga gcggatttga acgttgcgaa gcaacggccc ggagggtggc
     4981 gggcaggacg cccgccataa actgccagga attaattccc caggcatcaa ataaaacgaa
     5041 aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc
     5101 tgagtaggac aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg cccggagggt
     5161 ggcgggcagg acgcccgcca taaactgcca ggaattaatt ccccaggcat caaataaaac
     5221 gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc
     5281 tcctgagtag gacaaatccg ccgggagcgg atttgaacgt tgcgaagcaa cggcccggag
     5341 ggtggcgggc aggacgcccg ccataaactg ccaggaatta attccccagg catcaaataa
     5401 aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg
     5461 ctctcctgag taggacaaat ccgccgggag cggatttgaa cgttgcgaag caacggcccg
     5521 gagggtggcg ggcaggacgc ccgccataaa ctgccaggaa ttggggatcg gaattaattc
     5581 ccggtttaaa ccggggatct cgatcccgcg aaattaatac gactcactat aggggaattg
     5641 tgagcggata acaattcccc tctagaaata attttgttta actttaagaa ggagatatac
     5701 ttatgaaaat cgaagaaggt aaactggtaa tcggttccct ggagggttgc tttgccaagg
     5761 gtaccaatgt tttaatggcg gatgggtcta ttgaatgtat tgaaaacatt gaggttggta
     5821 ataaggtcat gggtaaagat ggcagacctc gtgaggtaat taaattgccc agaggaagag
     5881 aaactatgta cagcgtcgtg cagaaaagtc agcacagagc ccacaaaagt gactcaagtc
     5941 gtgaagtgcc agaattactc aagtttacgt gtaatgcgac ccatgagttg gttgttagaa
     6001 cacctcgtag tgtccgccgt ttgtctcgta ccattaaggg tgtcgaatat tttgaagtta
     6061 ttacttttga gatgggccaa aagaaagccc ccgacggtag aattgttgag cttgtcaagg
     6121 aagtttcaaa gagctaccca atatctgagg ggcctgagag agccaacgaa ttagtagaat
     6181 cctatagaaa ggcttcaaat aaagcttatt ttgagtggac tattgaggcc agagatcttt
     6241 ctctgttggg ttcccatgtt cgtaaagcta cctaccagac ttacgctcca attctttatg
     6301 agaatgacca ctttttcgac tacatgcaaa aaagtaagtt tcatctcacc attgaaggtc
     6361 caaaagtact tgcttattta cttggtttat ggattggtga tggattgtct gacagggcaa
     6421 ctttttcggt tgattccaga gatacttctt tgatggaacg tgttactgaa tatgctgaaa
     6481 agttgaattt gtgcgccgag tataaggaca gaaaagaacc acaagttgcc aaaactgtta
     6541 atttgtactc taaagttgtc agaggtgcta gcacaaatcc tggtgtatcc gcttggcagg
     6601 tcaacacagc ttatactgcg ggacaattgg tcacatataa cggcaagacg tataaatgtt
     6661 tgcagcccca cacctccttg gcaggatggg aaccatccaa cgttcctgcc ttgtggcagc
     6721 ttcaaggtgg ccacggtggt attcgcaata atcttaatac tgagaatcca ttatgggacg
     6781 ctattgttgg cttaggattc ttgaaggacg gtgtcaaaaa tattccttct ttcttgtcta
     6841 cggacaatat cggtactcgt gaaacatttc ttgctggtct aattgattct gatggctatg
     6901 ttactgatga gcatggtatt aaagcaacaa taaagacaat tcatacttct gtcagagatg
     6961 gtttggtttc ccttgctcgt tctttaggct tagtagtctc ggttaacgca gaacctgcta
     7021 aggttgacat gaatgtcacc aaacataaaa ttagttatgc tatttatatg tctggtggag
     7081 atgttttgct taacgttctt tcgaagtgtg ccggctctaa aaaattcagg cctgctcccg
     7141 ccgctgcttt tgcacgtgag tgccgcggat tttatttcga gttacaagaa ttgaaggaag
     7201 acgattatta tgggattact ttatctgatg attctgatca tcagtttttg cttggatctc
     7261 aggttgttgt acagaacatg gatccgaact gcagctgcgc ggcgggcgat agctgcacct
     7321 gcgcgggcag ctgcaaatgc aaagaatgca aatgcaccag ctgcaaaaaa agcctgagcg
     7381 atgaagattt taaagcggtg tttggcatga cccgtagcgc gtttgcgaac ctgccgctgt
     7441 ggctgcagca gcatctgctg aaagaaaaag gcctgtttta agatatcgtc gacggatccg
     7501 aattccctgc aggtaattaa ataactagtt gatccggctg ctaacaaagc ccgaaaggaa
     7561 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
     7621 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggataact acgtcaggtg
     7681 gcactttt
//
