LOCUS       sequence-203723-        7099 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmids for production of recombinant 
            metallothionein 4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7099)
  TITLE     Krezel Lab Plasmids
REFERENCE   2  (bases 1 to 7099)
  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"
COMMENT     Sequence Label: MT4
FEATURES             Location/Qualifiers
     source          1..7099
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      19..607
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     508..527
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     761..778
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(800..819)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     960..982
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(976..1167)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1724..2806)
                     /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     2768..2787
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2807..2884)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      3037..3123
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3220..3306
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3441..3460
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3441..3459
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3460..3484
                     /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             3499..3521
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3574..4392
                     /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             4399..4551
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVP
                     ALWQLQ"
     CDS             4555..5103
                     /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(5366..5384)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5380..5427
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        5487..5591
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5592..5660,5661..6452)
                     /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(5810..5829)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(6494..7007)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6689..6710)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6901..6920
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
       61 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
      121 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
      181 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
      241 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
      301 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
      361 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
      421 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
      481 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      541 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      601 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      661 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
      721 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
      781 agctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
      841 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
      901 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
      961 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     1021 gtaaagctca tcagcgtggt cgtgcagcga ttcacagatg tctgcctgtt catccgcgtc
     1081 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     1141 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     1201 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     1261 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     1321 ccagagaaaa atcactcagg gtcaatgcca gccgaacgcc agcaagacgt agcccagcgc
     1381 gtcggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
     1441 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
     1501 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
     1561 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
     1621 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     1681 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     1741 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     1801 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     1861 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     1921 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     1981 tatcgtcgta tcccactacc gagatgtccg caccaacgcg cagcccggac tcggtaatgg
     2041 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
     2101 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     2161 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
     2221 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
     2281 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
     2341 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
     2401 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     2461 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     2521 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     2581 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     2641 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     2701 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     2761 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     2821 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
     2881 tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt
     2941 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgccggca tgccgccctt
     3001 tcgtcttcaa gaattaattc ccaattcccc aggcatcaaa taaaacgaaa ggctcagtcg
     3061 aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca
     3121 aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg gcgggcagga
     3181 cgcccgccat aaactgccag gaattaattc cccaggcatc aaataaaacg aaaggctcag
     3241 tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg
     3301 acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca
     3361 ggacgcccgc cataaactgc caggaattgg ggatcggaat taattcccgg tttaaaccgg
     3421 ggatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
     3481 ttcccctcta gaaataattt tgtttaactt taagaaggag atatacttat gaaaatcgaa
     3541 gaaggtaaac tggtaatcgg ttccctggag ggttgctttg ccaagggtac caatgtttta
     3601 atggcggatg ggtctattga atgtattgaa aacattgagg ttggtaataa ggtcatgggt
     3661 aaagatggca gacctcgtga ggtaattaaa ttgcccagag gaagagaaac tatgtacagc
     3721 gtcgtgcaga aaagtcagca cagagcccac aaaagtgact caagtcgtga agtgccagaa
     3781 ttactcaagt ttacgtgtaa tgcgacccat gagttggttg ttagaacacc tcgtagtgtc
     3841 cgccgtttgt ctcgtaccat taagggtgtc gaatattttg aagttattac ttttgagatg
     3901 ggccaaaaga aagcccccga cggtagaatt gttgagcttg tcaaggaagt ttcaaagagc
     3961 tacccaatat ctgaggggcc tgagagagcc aacgaattag tagaatccta tagaaaggct
     4021 tcaaataaag cttattttga gtggactatt gaggccagag atctttctct gttgggttcc
     4081 catgttcgta aagctaccta ccagacttac gctccaattc tttatgagaa tgaccacttt
     4141 ttcgactaca tgcaaaaaag taagtttcat ctcaccattg aaggtccaaa agtacttgct
     4201 tatttacttg gtttatggat tggtgatgga ttgtctgaca gggcaacttt ttcggttgat
     4261 tccagagata cttctttgat ggaacgtgtt actgaatatg ctgaaaagtt gaatttgtgc
     4321 gccgagtata aggacagaaa agaaccacaa gttgccaaaa ctgttaattt gtactctaaa
     4381 gttgtcagag gtgctagcac aaatcctggt gtatccgctt ggcaggtcaa cacagcttat
     4441 actgcgggac aattggtcac atataacggc aagacgtata aatgtttgca gccccacacc
     4501 tccttggcag gatgggaacc atccaacgtt cctgccttgt ggcagcttca aggtggccac
     4561 ggtggtattc gcaataatct taatactgag aatccattat gggacgctat tgttggctta
     4621 ggattcttga aggacggtgt caaaaatatt ccttctttct tgtctacgga caatatcggt
     4681 actcgtgaaa catttcttgc tggtctaatt gattctgatg gctatgttac tgatgagcat
     4741 ggtattaaag caacaataaa gacaattcat acttctgtca gagatggttt ggtttccctt
     4801 gctcgttctt taggcttagt agtctcggtt aacgcagaac ctgctaaggt tgacatgaat
     4861 gtcaccaaac ataaaattag ttatgctatt tatatgtctg gtggagatgt tttgcttaac
     4921 gttctttcga agtgtgccgg ctctaaaaaa ttcaggcctg ctcccgccgc tgcttttgca
     4981 cgtgagtgcc gcggatttta tttcgagtta caagaattga aggaagacga ttattatggg
     5041 attactttat ctgatgattc tgatcatcag tttttgcttg gatctcaggt tgttgtacag
     5101 aacatggacc cgcgtgaatg tgtctgtatg agtggtggta tttgtatgtg tggtgataac
     5161 tgtaagtgta cgacgtgtaa ctgtaaaacg tgccgcaaaa gttgctgtcc gtgctgtccg
     5221 ccgggttgcg caaagtgtgc ccgtggttgc atttgtaaag gcggtagcga taagtgctct
     5281 tgctgtccgt aactgcaggt aattaaataa ctagttgatc cggctgctaa caaagcccga
     5341 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     5401 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg ataactacgt
     5461 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     5521 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     5581 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     5641 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     5701 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     5761 gttttcgccc cgaagaacgt ttcccaatga tgagcacttt taaagttctg ctatgtggcg
     5821 cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     5881 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     5941 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     6001 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     6061 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     6121 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     6181 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     6241 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     6301 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     6361 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     6421 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     6481 tttagattga tttaccccgg ttgataatca gaaaagcccc aaaaacagga agattgtata
     6541 agcaaatatt taaattgtaa acgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     6601 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     6661 atagcccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     6721 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     6781 accatcaccc aaatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     6841 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     6901 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     6961 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtaaa aggatctagg
     7021 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     7081 gagcgtcaga ccccgtaga
//
