LOCUS       sequence-203320-        7096 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmids for production of recombinant 
            metallothionein 1b.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7096)
  TITLE     Krezel Lab Plasmids
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"
COMMENT     Sequence Label: MT1B
FEATURES             Location/Qualifiers
     source          1..7096
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        8..112
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(113..181,182..973)
                     /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(331..350)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(1015..1528)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1210..1231)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1422..1441
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      1639..2227
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2128..2147
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2381..2398
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2420..2439)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2580..2602
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2596..2787)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3344..4426)
                     /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     4388..4407
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4427..4504)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      4657..4743
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4840..4926
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     5061..5080
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5061..5079
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5080..5104
                     /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             5119..5141
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5194..6012
                     /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             6019..6171
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVP
                     ALWQLQ"
     CDS             6175..6723
                     /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(6983..7001)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6997..7044
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
       61 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
      121 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
      181 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
      241 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
      301 tttcccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga
      361 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
      421 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
      481 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
      541 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
      601 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
      661 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
      721 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
      781 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
      841 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
      901 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
      961 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaccccg
     1021 gttgataatc agaaaagccc caaaaacagg aagattgtat aagcaaatat ttaaattgta
     1081 aacgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     1141 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagcccga gatagggttg
     1201 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     1261 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc caaatcaagt
     1321 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     1381 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     1441 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     1501 gcgcttaatg cgccgctaca gggcgcgtaa aaggatctag gtgaagatcc tttttgataa
     1561 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     1621 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     1681 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     1741 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     1801 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     1861 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     1921 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     1981 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     2041 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     2101 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     2161 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     2221 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     2281 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     2341 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     2401 agctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     2461 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     2521 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     2581 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     2641 gtaaagctca tcagcgtggt cgtgcagcga ttcacagatg tctgcctgtt catccgcgtc
     2701 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     2761 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     2821 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     2881 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     2941 ccagagaaaa atcactcagg gtcaatgcca gccgaacgcc agcaagacgt agcccagcgc
     3001 gtcggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
     3061 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
     3121 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
     3181 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
     3241 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     3301 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     3361 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     3421 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     3481 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     3541 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     3601 tatcgtcgta tcccactacc gagatgtccg caccaacgcg cagcccggac tcggtaatgg
     3661 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
     3721 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     3781 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
     3841 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
     3901 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
     3961 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
     4021 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     4081 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     4141 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     4201 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     4261 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     4321 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     4381 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     4441 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
     4501 tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt
     4561 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgccggca tgccgccctt
     4621 tcgtcttcaa gaattaattc ccaattcccc aggcatcaaa taaaacgaaa ggctcagtcg
     4681 aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca
     4741 aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg gcgggcagga
     4801 cgcccgccat aaactgccag gaattaattc cccaggcatc aaataaaacg aaaggctcag
     4861 tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg
     4921 acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca
     4981 ggacgcccgc cataaactgc caggaattgg ggatcggaat taattcccgg tttaaaccgg
     5041 ggatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
     5101 ttcccctcta gaaataattt tgtttaactt taagaaggag atatacttat gaaaatcgaa
     5161 gaaggtaaac tggtaatcgg ttccctggag ggttgctttg ccaagggtac caatgtttta
     5221 atggcggatg ggtctattga atgtattgaa aacattgagg ttggtaataa ggtcatgggt
     5281 aaagatggca gacctcgtga ggtaattaaa ttgcccagag gaagagaaac tatgtacagc
     5341 gtcgtgcaga aaagtcagca cagagcccac aaaagtgact caagtcgtga agtgccagaa
     5401 ttactcaagt ttacgtgtaa tgcgacccat gagttggttg ttagaacacc tcgtagtgtc
     5461 cgccgtttgt ctcgtaccat taagggtgtc gaatattttg aagttattac ttttgagatg
     5521 ggccaaaaga aagcccccga cggtagaatt gttgagcttg tcaaggaagt ttcaaagagc
     5581 tacccaatat ctgaggggcc tgagagagcc aacgaattag tagaatccta tagaaaggct
     5641 tcaaataaag cttattttga gtggactatt gaggccagag atctttctct gttgggttcc
     5701 catgttcgta aagctaccta ccagacttac gctccaattc tttatgagaa tgaccacttt
     5761 ttcgactaca tgcaaaaaag taagtttcat ctcaccattg aaggtccaaa agtacttgct
     5821 tatttacttg gtttatggat tggtgatgga ttgtctgaca gggcaacttt ttcggttgat
     5881 tccagagata cttctttgat ggaacgtgtt actgaatatg ctgaaaagtt gaatttgtgc
     5941 gccgagtata aggacagaaa agaaccacaa gttgccaaaa ctgttaattt gtactctaaa
     6001 gttgtcagag gtgctagcac aaatcctggt gtatccgctt ggcaggtcaa cacagcttat
     6061 actgcgggac aattggtcac atataacggc aagacgtata aatgtttgca gccccacacc
     6121 tccttggcag gatgggaacc atccaacgtt cctgccttgt ggcagcttca aggtggccac
     6181 ggtggtattc gcaataatct taatactgag aatccattat gggacgctat tgttggctta
     6241 ggattcttga aggacggtgt caaaaatatt ccttctttct tgtctacgga caatatcggt
     6301 actcgtgaaa catttcttgc tggtctaatt gattctgatg gctatgttac tgatgagcat
     6361 ggtattaaag caacaataaa gacaattcat acttctgtca gagatggttt ggtttccctt
     6421 gctcgttctt taggcttagt agtctcggtt aacgcagaac ctgctaaggt tgacatgaat
     6481 gtcaccaaac ataaaattag ttatgctatt tatatgtctg gtggagatgt tttgcttaac
     6541 gttctttcga agtgtgccgg ctctaaaaaa ttcaggcctg ctcccgccgc tgcttttgca
     6601 cgtgagtgcc gcggatttta tttcgagtta caagaattga aggaagacga ttattatggg
     6661 attactttat ctgatgattc tgatcatcag tttttgcttg gatctcaggt tgttgtacag
     6721 aacatggatc cgaattgctc atgtacgacg ggtggcagtt gtgcttgtgc tggctcctgt
     6781 aaatgtaaag aatgtaaatg tacgagttgt aaaaagtgct gttgcagttg ttgcccggtg
     6841 ggttgtgcaa aatgcgcaca gggctgtgtt tgcaaaggta gctctgaaaa gtgtcgttgt
     6901 tgcgcgtaac tgcaggtaat taaataacta gttgatccgg ctgctaacaa agcccgaaag
     6961 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     7021 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggata actacgtcag
     7081 gtggcacttt tcgggg
//
