LOCUS       sequence-203329-        7096 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmids for production of recombinant 
            metallothionein 1x.
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: MT1X
FEATURES             Location/Qualifiers
     source          1..7096
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        3..107
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(108..176,177..968)
                     /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(326..345)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(1010..1523)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1205..1226)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1417..1436
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      1634..2222
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2123..2142
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2376..2393
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2415..2434)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2575..2597
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2591..2782)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3339..4421)
                     /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     4383..4402
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4422..4499)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      4652..4738
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4835..4921
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     5056..5075
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5056..5074
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5075..5099
                     /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             5114..5136
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5189..6007
                     /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             6014..6166
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVP
                     ALWQLQ"
     CDS             6170..6718
                     /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(6978..6996)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6992..7039
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
       61 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
      121 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
      181 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
      241 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgtttcc
      301 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg
      361 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
      421 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
      481 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
      541 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
      601 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
      661 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
      721 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
      781 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
      841 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
      901 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
      961 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta ccccggttga
     1021 taatcagaaa agccccaaaa acaggaagat tgtataagca aatatttaaa ttgtaaacgt
     1081 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     1141 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag cccgagatag ggttgagtgt
     1201 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     1261 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcacccaaat caagtttttt
     1321 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     1381 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     1441 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     1501 taatgcgccg ctacagggcg cgtaaaagga tctaggtgaa gatccttttt gataatctca
     1561 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     1621 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     1681 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     1741 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     1801 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     1861 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     1921 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     1981 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     2041 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     2101 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     2161 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     2221 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     2281 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     2341 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcta
     2401 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     2461 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     2521 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     2581 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa
     2641 gctcatcagc gtggtcgtgc agcgattcac agatgtctgc ctgttcatcc gcgtccagct
     2701 cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg
     2761 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg
     2821 taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg
     2881 cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga
     2941 gaaaaatcac tcagggtcaa tgccagccga acgccagcaa gacgtagccc agcgcgtcgg
     3001 ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga
     3061 cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg
     3121 tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc
     3181 ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg
     3241 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     3301 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     3361 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     3421 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     3481 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     3541 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     3601 tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt aatggcgcgc
     3661 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     3721 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     3781 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     3841 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     3901 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     3961 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     4021 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     4081 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     4141 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     4201 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     4261 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     4321 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     4381 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     4441 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
     4501 gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt
     4561 gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc cggcatgccg ccctttcgtc
     4621 ttcaagaatt aattcccaat tccccaggca tcaaataaaa cgaaaggctc agtcgaaaga
     4681 ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc
     4741 gccgggagcg gatttgaacg ttgcgaagca acggcccgga gggtggcggg caggacgccc
     4801 gccataaact gccaggaatt aattccccag gcatcaaata aaacgaaagg ctcagtcgaa
     4861 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa
     4921 tccgccggga gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg
     4981 cccgccataa actgccagga attggggatc ggaattaatt cccggtttaa accggggatc
     5041 tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc
     5101 ctctagaaat aattttgttt aactttaaga aggagatata cttatgaaaa tcgaagaagg
     5161 taaactggta atcggttccc tggagggttg ctttgccaag ggtaccaatg ttttaatggc
     5221 ggatgggtct attgaatgta ttgaaaacat tgaggttggt aataaggtca tgggtaaaga
     5281 tggcagacct cgtgaggtaa ttaaattgcc cagaggaaga gaaactatgt acagcgtcgt
     5341 gcagaaaagt cagcacagag cccacaaaag tgactcaagt cgtgaagtgc cagaattact
     5401 caagtttacg tgtaatgcga cccatgagtt ggttgttaga acacctcgta gtgtccgccg
     5461 tttgtctcgt accattaagg gtgtcgaata ttttgaagtt attacttttg agatgggcca
     5521 aaagaaagcc cccgacggta gaattgttga gcttgtcaag gaagtttcaa agagctaccc
     5581 aatatctgag gggcctgaga gagccaacga attagtagaa tcctatagaa aggcttcaaa
     5641 taaagcttat tttgagtgga ctattgaggc cagagatctt tctctgttgg gttcccatgt
     5701 tcgtaaagct acctaccaga cttacgctcc aattctttat gagaatgacc actttttcga
     5761 ctacatgcaa aaaagtaagt ttcatctcac cattgaaggt ccaaaagtac ttgcttattt
     5821 acttggttta tggattggtg atggattgtc tgacagggca actttttcgg ttgattccag
     5881 agatacttct ttgatggaac gtgttactga atatgctgaa aagttgaatt tgtgcgccga
     5941 gtataaggac agaaaagaac cacaagttgc caaaactgtt aatttgtact ctaaagttgt
     6001 cagaggtgct agcacaaatc ctggtgtatc cgcttggcag gtcaacacag cttatactgc
     6061 gggacaattg gtcacatata acggcaagac gtataaatgt ttgcagcccc acacctcctt
     6121 ggcaggatgg gaaccatcca acgttcctgc cttgtggcag cttcaaggtg gccacggtgg
     6181 tattcgcaat aatcttaata ctgagaatcc attatgggac gctattgttg gcttaggatt
     6241 cttgaaggac ggtgtcaaaa atattccttc tttcttgtct acggacaata tcggtactcg
     6301 tgaaacattt cttgctggtc taattgattc tgatggctat gttactgatg agcatggtat
     6361 taaagcaaca ataaagacaa ttcatacttc tgtcagagat ggtttggttt cccttgctcg
     6421 ttctttaggc ttagtagtct cggttaacgc agaacctgct aaggttgaca tgaatgtcac
     6481 caaacataaa attagttatg ctatttatat gtctggtgga gatgttttgc ttaacgttct
     6541 ttcgaagtgt gccggctcta aaaaattcag gcctgctccc gccgctgctt ttgcacgtga
     6601 gtgccgcgga ttttatttcg agttacaaga attgaaggaa gacgattatt atgggattac
     6661 tttatctgat gattctgatc atcagttttt gcttggatct caggttgttg tacagaacat
     6721 ggacccgaat tgtagctgtt cgccggtggg ctcctgtgcc tgtgctggct cctgtaaatg
     6781 taaagaatgt aaatgtacct cgtgcaaaaa gagctgctgt tcttgctgtc cggtgggttg
     6841 cgcaaaatgt gcacagggtt gcatttgtaa aggtaccagt gataagtgca gctgctgtgc
     6901 gtaactgcag gtaattaaat aactagttga tccggctgct aacaaagccc gaaaggaagc
     6961 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     7021 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggataactac gtcaggtggc
     7081 acttttcggg gaaatg
//
