LOCUS       sequence-394917-        7634 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmids for production of recombinant Blue 
            Crab Callinectes sapidus Metallothionein 1B.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7634)
  AUTHORS   Mosna K, Jurczak K, Krezel A
  TITLE     Differentiated Zn(II) binding affinities in animal, plant, and 
            bacterial metallothioneins define their zinc buffering capacity at 
            physiological pZn.
  JOURNAL   Metallomics. 2023 Oct 4;15(10):mfad061. doi: 10.1093/mtomcs/mfad061.
  PUBMED    37804185
REFERENCE   2  (bases 1 to 7634)
  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: "2023-10-4"; volume: "15(10):mfad061. doi"; 
            pages: " 10.1093/mtomcs/mfad061"
COMMENT     Sequence Label: BcrMT1B Blue Crab Metallothionein 1B
FEATURES             Location/Qualifiers
     source          1..7634
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(102..170,171..962)
                     /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(320..339)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(1004..1517)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1199..1220)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1411..1430
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      1628..2216
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2117..2136
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2370..2387
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2409..2428)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2569..2591
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2585..2776)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3333..4415)
                     /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     4377..4396
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4416..4493)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      4646..4732
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4829..4915
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5012..5098
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5195..5281
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5378..5464
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     5599..5618
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5599..5617
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5618..5642
                     /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             5657..5679
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5732..6550
                     /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             6557..6709
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVP
                     ALWQLQ"
     CDS             6713..7261
                     /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(7510..7528)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7524..7571
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        join(7631..7634,1..101)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
       61 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
      121 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
      181 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
      241 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt ttcccaatga
      301 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag
      361 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
      421 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
      481 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
      541 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
      601 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
      661 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
      721 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
      781 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
      841 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
      901 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
      961 aactgtcaga ccaagtttac tcatatatac tttagattga tttaccccgg ttgataatca
     1021 gaaaagcccc aaaaacagga agattgtata agcaaatatt taaattgtaa acgttaatat
     1081 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     1141 aatcggcaaa atcccttata aatcaaaaga atagcccgag atagggttga gtgttgttcc
     1201 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     1261 cgtctatcag ggcgatggcc cactacgtga accatcaccc aaatcaagtt ttttggggtc
     1321 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     1381 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     1441 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     1501 gccgctacag ggcgcgtaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     1561 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     1621 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     1681 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     1741 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     1801 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     1861 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     1921 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     1981 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     2041 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     2101 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     2161 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     2221 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     2281 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     2341 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gctatggtgc
     2401 actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
     2461 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac
     2521 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     2581 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
     2641 cagcgtggtc gtgcagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
     2701 gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
     2761 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
     2821 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
     2881 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
     2941 tcactcaggg tcaatgccag ccgaacgcca gcaagacgta gcccagcgcg tcggccgcca
     3001 tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg
     3061 cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc
     3121 tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga
     3181 gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc
     3241 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat
     3301 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     3361 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     3421 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
     3481 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
     3541 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
     3601 cccactaccg agatgtccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
     3661 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc
     3721 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
     3781 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
     3841 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
     3901 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
     3961 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
     4021 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
     4081 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
     4141 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
     4201 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
     4261 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
     4321 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
     4381 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
     4441 tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc
     4501 tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc
     4561 gttgagcacc gccgccgcaa ggaatggtgc atgccggcat gccgcccttt cgtcttcaag
     4621 aattaattcc caattcccca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc
     4681 ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccggg
     4741 agcggatttg aacgttgcga agcaacggcc cggagggtgg cgggcaggac gcccgccata
     4801 aactgccagg aattaattcc ccaggcatca aataaaacga aaggctcagt cgaaagactg
     4861 ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc
     4921 gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc
     4981 ataaactgcc aggaattaat tccccaggca tcaaataaaa cgaaaggctc agtcgaaaga
     5041 ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc
     5101 gccgggagcg gatttgaacg ttgcgaagca acggcccgga gggtggcggg caggacgccc
     5161 gccataaact gccaggaatt aattccccag gcatcaaata aaacgaaagg ctcagtcgaa
     5221 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa
     5281 tccgccggga gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg
     5341 cccgccataa actgccagga attaattccc caggcatcaa ataaaacgaa aggctcagtc
     5401 gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac
     5461 aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg
     5521 acgcccgcca taaactgcca ggaattgggg atcggaatta attcccggtt taaaccgggg
     5581 atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt
     5641 cccctctaga aataattttg tttaacttta agaaggagat atacttatga aaatcgaaga
     5701 aggtaaactg gtaatcggtt ccctggaggg ttgctttgcc aagggtacca atgttttaat
     5761 ggcggatggg tctattgaat gtattgaaaa cattgaggtt ggtaataagg tcatgggtaa
     5821 agatggcaga cctcgtgagg taattaaatt gcccagagga agagaaacta tgtacagcgt
     5881 cgtgcagaaa agtcagcaca gagcccacaa aagtgactca agtcgtgaag tgccagaatt
     5941 actcaagttt acgtgtaatg cgacccatga gttggttgtt agaacacctc gtagtgtccg
     6001 ccgtttgtct cgtaccatta agggtgtcga atattttgaa gttattactt ttgagatggg
     6061 ccaaaagaaa gcccccgacg gtagaattgt tgagcttgtc aaggaagttt caaagagcta
     6121 cccaatatct gaggggcctg agagagccaa cgaattagta gaatcctata gaaaggcttc
     6181 aaataaagct tattttgagt ggactattga ggccagagat ctttctctgt tgggttccca
     6241 tgttcgtaaa gctacctacc agacttacgc tccaattctt tatgagaatg accacttttt
     6301 cgactacatg caaaaaagta agtttcatct caccattgaa ggtccaaaag tacttgctta
     6361 tttacttggt ttatggattg gtgatggatt gtctgacagg gcaacttttt cggttgattc
     6421 cagagatact tctttgatgg aacgtgttac tgaatatgct gaaaagttga atttgtgcgc
     6481 cgagtataag gacagaaaag aaccacaagt tgccaaaact gttaatttgt actctaaagt
     6541 tgtcagaggt gctagcacaa atcctggtgt atccgcttgg caggtcaaca cagcttatac
     6601 tgcgggacaa ttggtcacat ataacggcaa gacgtataaa tgtttgcagc cccacacctc
     6661 cttggcagga tgggaaccat ccaacgttcc tgccttgtgg cagcttcaag gtggccacgg
     6721 tggtattcgc aataatctta atactgagaa tccattatgg gacgctattg ttggcttagg
     6781 attcttgaag gacggtgtca aaaatattcc ttctttcttg tctacggaca atatcggtac
     6841 tcgtgaaaca tttcttgctg gtctaattga ttctgatggc tatgttactg atgagcatgg
     6901 tattaaagca acaataaaga caattcatac ttctgtcaga gatggtttgg tttcccttgc
     6961 tcgttcttta ggcttagtag tctcggttaa cgcagaacct gctaaggttg acatgaatgt
     7021 caccaaacat aaaattagtt atgctattta tatgtctggt ggagatgttt tgcttaacgt
     7081 tctttcgaag tgtgccggct ctaaaaaatt caggcctgct cccgccgctg cttttgcacg
     7141 tgagtgccgc ggattttatt tcgagttaca agaattgaag gaagacgatt attatgggat
     7201 tactttatct gatgattctg atcatcagtt tttgcttgga tctcaggttg ttgtacagaa
     7261 catgccgggt ccgtgctgta atgataaatg cgtgtgccaa gaaggcggtt gtaaagcggg
     7321 ctgccaatgt acctcgtgtc gttgttcgcc gtgccagaaa tgtaccagtg gctgcaaatg
     7381 tgcgacgaaa gaagaatgca gcaaaacctg tacgaaaccg tgctcttgct gtccgaaata
     7441 aggtaattaa ataactagtt gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
     7501 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     7561 ggggtttttt gctgaaagga ggaactatat ccggataact acgtcaggtg gcacttttcg
     7621 gggaaatgtg cgcg
//
