LOCUS       sequence-203321-        7096 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmids for production of recombinant 
            metallothionein 1e.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7096)
  AUTHORS   Peris-Diaz MD, Guran R, Zitka O, Adam V, Krezel A
  TITLE     Mass Spectrometry-Based Structural Analysis of Cysteine-Rich 
            Metal-Binding Sites in Proteins with MetaOdysseus R Software.
  JOURNAL   J Proteome Res. 2021 Jan 1;20(1):776-785. doi: 
            10.1021/acs.jproteome.0c00651. Epub 2020 Sep 28.
  PUBMED    32924499
REFERENCE   2  (bases 1 to 7096)
  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"; doi: 
            "10.1021/acs.jproteome.0c00651"; journalName: "J Proteome Res"; 
            date: "2021-01-1- 1"; volume: "20"; issue: "1"; pages: "776-785"
COMMENT     Sequence Label: MT1E
FEATURES             Location/Qualifiers
     source          1..7096
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      145..231
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     366..385
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        366..384
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    385..409
                     /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             424..446
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             499..1317
                     /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             1324..1476
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVP
                     ALWQLQ"
     CDS             1480..2028
                     /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(2288..2306)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2302..2349
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        2409..2513
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2514..2582,2583..3374)
                     /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(2732..2751)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(3416..3929)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3611..3632)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3823..3842
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      4040..4628
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4529..4548
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4782..4799
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(4821..4840)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4981..5003
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4997..5188)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5745..6827)
                     /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     6789..6808
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6828..6905)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      join(7058..7096,1..48)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 cgttttatct gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg
       61 gatttgaacg ttgcgaagca acggcccgga gggtggcggg caggacgccc gccataaact
      121 gccaggaatt aattccccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc
      181 tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccggga
      241 gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa
      301 actgccagga attggggatc ggaattaatt cccggtttaa accggggatc tcgatcccgc
      361 gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat
      421 aattttgttt aactttaaga aggagatata cttatgaaaa tcgaagaagg taaactggta
      481 atcggttccc tggagggttg ctttgccaag ggtaccaatg ttttaatggc ggatgggtct
      541 attgaatgta ttgaaaacat tgaggttggt aataaggtca tgggtaaaga tggcagacct
      601 cgtgaggtaa ttaaattgcc cagaggaaga gaaactatgt acagcgtcgt gcagaaaagt
      661 cagcacagag cccacaaaag tgactcaagt cgtgaagtgc cagaattact caagtttacg
      721 tgtaatgcga cccatgagtt ggttgttaga acacctcgta gtgtccgccg tttgtctcgt
      781 accattaagg gtgtcgaata ttttgaagtt attacttttg agatgggcca aaagaaagcc
      841 cccgacggta gaattgttga gcttgtcaag gaagtttcaa agagctaccc aatatctgag
      901 gggcctgaga gagccaacga attagtagaa tcctatagaa aggcttcaaa taaagcttat
      961 tttgagtgga ctattgaggc cagagatctt tctctgttgg gttcccatgt tcgtaaagct
     1021 acctaccaga cttacgctcc aattctttat gagaatgacc actttttcga ctacatgcaa
     1081 aaaagtaagt ttcatctcac cattgaaggt ccaaaagtac ttgcttattt acttggttta
     1141 tggattggtg atggattgtc tgacagggca actttttcgg ttgattccag agatacttct
     1201 ttgatggaac gtgttactga atatgctgaa aagttgaatt tgtgcgccga gtataaggac
     1261 agaaaagaac cacaagttgc caaaactgtt aatttgtact ctaaagttgt cagaggtgct
     1321 agcacaaatc ctggtgtatc cgcttggcag gtcaacacag cttatactgc gggacaattg
     1381 gtcacatata acggcaagac gtataaatgt ttgcagcccc acacctcctt ggcaggatgg
     1441 gaaccatcca acgttcctgc cttgtggcag cttcaaggtg gccacggtgg tattcgcaat
     1501 aatcttaata ctgagaatcc attatgggac gctattgttg gcttaggatt cttgaaggac
     1561 ggtgtcaaaa atattccttc tttcttgtct acggacaata tcggtactcg tgaaacattt
     1621 cttgctggtc taattgattc tgatggctat gttactgatg agcatggtat taaagcaaca
     1681 ataaagacaa ttcatacttc tgtcagagat ggtttggttt cccttgctcg ttctttaggc
     1741 ttagtagtct cggttaacgc agaacctgct aaggttgaca tgaatgtcac caaacataaa
     1801 attagttatg ctatttatat gtctggtgga gatgttttgc ttaacgttct ttcgaagtgt
     1861 gccggctcta aaaaattcag gcctgctccc gccgctgctt ttgcacgtga gtgccgcgga
     1921 ttttatttcg agttacaaga attgaaggaa gacgattatt atgggattac tttatctgat
     1981 gattctgatc atcagttttt gcttggatct caggttgttg tacagaacat ggacccgaat
     2041 tgctcatgtg ctacgggcgg ttcctgtacc tgtgctggct cctgtaaatg taaagaatgt
     2101 aaatgtacct cgtgtaaaaa gagctgctgt tcttgctgtc cggtgggttg cgcaaaatgt
     2161 gcacagggtt gcgtttgtaa aggtgcaagt gaaaagtgca gctgctgtgc gtaactgcag
     2221 gtaattaaat aactagttga tccggctgct aacaaagccc gaaaggaagc tgagttggct
     2281 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     2341 ggttttttgc tgaaaggagg aactatatcc ggataactac gtcaggtggc acttttcggg
     2401 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     2461 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     2521 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     2581 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     2641 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     2701 gtttcccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg
     2761 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     2821 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     2881 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     2941 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     3001 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     3061 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     3121 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     3181 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     3241 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     3301 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     3361 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttacccc
     3421 ggttgataat cagaaaagcc ccaaaaacag gaagattgta taagcaaata tttaaattgt
     3481 aaacgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     3541 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagcccg agatagggtt
     3601 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     3661 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac ccaaatcaag
     3721 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     3781 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     3841 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     3901 cgcgcttaat gcgccgctac agggcgcgta aaaggatcta ggtgaagatc ctttttgata
     3961 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     4021 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     4081 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     4141 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     4201 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     4261 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     4321 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     4381 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     4441 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     4501 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     4561 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     4621 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     4681 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     4741 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     4801 aagctatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
     4861 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
     4921 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     4981 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
     5041 ggtaaagctc atcagcgtgg tcgtgcagcg attcacagat gtctgcctgt tcatccgcgt
     5101 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
     5161 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
     5221 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
     5281 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
     5341 accagagaaa aatcactcag ggtcaatgcc agccgaacgc cagcaagacg tagcccagcg
     5401 cgtcggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac
     5461 cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga
     5521 tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca
     5581 cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc
     5641 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
     5701 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     5761 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     5821 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     5881 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     5941 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     6001 gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg
     6061 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     6121 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     6181 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     6241 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     6301 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     6361 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     6421 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     6481 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     6541 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     6601 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     6661 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     6721 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     6781 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     6841 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     6901 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     6961 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgccggc atgccgccct
     7021 ttcgtcttca agaattaatt cccaattccc caggcatcaa ataaaacgaa aggctcagtc
     7081 gaaagactgg gccttt
//
