LOCUS       sequence-468896-        7872 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for bacterial expression and purification of DjCas13d 
            protein (zebrafish codon-optimized).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7872)
  AUTHORS   Moreno-Sanchez I, Hernandez-Huertas L, Nahon-Cano D, Martinez-Garcia
            PM, Treichel AJ, Gomez-Marin C, Tomas-Gallardo L, da Silva Pescador 
            G, Kushawah G, Egidy R, Perera A, Diaz-Moscoso A, Cano-Ruiz A, 
            Walker JA 2nd, Munoz MJ, Holden K, Galceran J, Nieto MA, Bazzini AA,
            Moreno-Mateos MA
  TITLE     Enhanced RNA-targeting CRISPR-Cas technology in zebrafish.
  JOURNAL   Nat Commun. 2025 Mar 16;16(1):2591. doi: 10.1038/s41467-025-57792-9.
  PUBMED    40091120
REFERENCE   2  (bases 1 to 7872)
  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: "Nat 
            Commun."; date: "2025-03-16"; volume: "16(1):2591. doi"; pages: " 
            10.1038/s41467-025-57792-9"
COMMENT     Sequence Label: pET28b-zDjCas13d-His
FEATURES             Location/Qualifiers
     source          1..7872
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    5..144
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(70..89)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     230..252
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(246..437)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1246..2328)
                     /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     2290..2309
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2329..2406)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2612..2631
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2715..2734
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2715..2733
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2734..2758
                     /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             2773..2795
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5451..5468
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5521..5539)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5535..5582
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5619..6074
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     5916..5937
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6166..6981)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(6166..6981)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     6889..6908
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6973..6992
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      7103..7691
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7592..7611
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7845..7862
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
       61 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
      121 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
      181 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
      241 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
      301 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
      361 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
      421 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
      481 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
      541 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
      601 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
      661 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
      721 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
      781 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
      841 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
      901 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
      961 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg
     1021 atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc
     1081 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg
     1141 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     1201 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     1261 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     1321 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     1381 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     1441 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     1501 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
     1561 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     1621 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     1681 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     1741 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     1801 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     1861 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     1921 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     1981 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     2041 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     2101 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     2161 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     2221 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     2281 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     2341 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     2401 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     2461 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     2521 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     2581 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     2641 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
     2701 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
     2761 tctagaaata attttgttta actttaagaa ggagatatac catggcaaag aagcagaagt
     2821 ccaagaagac cgtgtccaag accagcggcc tgaaggaagc cctgagcgtc cagggcaccg
     2881 tgatcatgac cagcttcggc aagggcaaca tggctaacct gagctacaag attcccagca
     2941 gccagaagcc tcagaacctg aacagctccg ccggcctgaa gaacgtggag gtgagcggca
     3001 agaagatcaa gttccagggc cgtcacccca aaatcgccac caccgacaac cccctgttca
     3061 agccccagcc cggcatggac ctgctgtgcc tgaaggacaa gctggaaatg cactacttcg
     3121 gtaagacctt cgacgacaac atccacatcc agctgatcta ccagatcctg gatatcgaga
     3181 agatcctggc cgtgcatgtg aacaacatcg tgttcaccct ggacaacgtg ctgcaccccc
     3241 agaaggagga gctgaccgaa gatttcatcg gcgccggcgg ctggaggatc aatcttgact
     3301 accagaccct gagaggacag accaataagt atgaccgttt taagaattac atcaagcgaa
     3361 aggagctgct gtacttcggc gaagccttct accacgagaa cgaacgtagg tacgaggaag
     3421 acatcttcgc tattctgacc ctgctgagcg ccctgcgtca gttctgcttc cactccgacc
     3481 ttagcagcga tgagtctgac catgtgaact cgttctggct gtaccagctg gaggatcagc
     3541 tgagcgacga gttcaaggaa accctgagca tcctgtggga ggaggttacc gagaggatcg
     3601 acagcgagtt cctgaagacc aacaccgtga atctgcacat cctgtgccac gtgttcccga
     3661 aggagagcaa ggaaaccata gtgagggcct actacgagtt cctgatcaaa aagagcttca
     3721 agaacatggg ctttagcatc aaaaagctgc gtgagatcat gctggaacag agcgacctga
     3781 agagcttcaa ggaggacaag tacaattctg tcagggccaa gctgtacaag ctgttcgact
     3841 tcatcatcac atactactac gatcaccacg ccttcgagaa ggaggccctg gtgagctccc
     3901 tgaggagcag cctgaccgag gaaaacaagg aggagatcta catcaagaca gcccgtacac
     3961 tggccagcgc cctgggcgcc gacttcaaga aggccgccgc cgatgtgaac gctaagaaca
     4021 tcagggacta ccagaagaag gcaaacgatt accgtattag cttcgaggac atcaagatcg
     4081 gaaataccgg catcggctac ttcagcgagc tgatctatat gctgaccctg ctgctggacg
     4141 gcaaggagat caacgacctg ctgaccaccc tgatcaacaa gttcgacaac attatctcct
     4201 tcatcgacat acttaagaag ctgaacctgg agttcaaatt caagcctgag tacgccgatt
     4261 tcttcaatat gaccaactgc cgttacaccc tggaggaact gagggtgatc aatagcatcg
     4321 ccaggatgca gaagcccagc gccgacgccc gtaagatcat gtaccgtgac gccctgcgga
     4381 tcctgggcat ggacaacagg cccgacgagg aaatcgaccg tgagctggag cgaaccatgc
     4441 ccgtgggcgc cgacggcaag ttcatcaagg gcaagcaggg attcaggaac tttatcgcca
     4501 gcaacgtgat cgagagcagc aggttccatt acctggtgcg ttacaacaat ccacacaaga
     4561 cacgaaccct ggtgaagaat cctaacgtgg tgaagttcgt gctggaggga atccctgaga
     4621 cccagataaa gcgttacttc gacgtctgca agggccagga gatccccccc accagcgaca
     4681 agagcgctca gatcgacgtg ctggccagaa tcatcagcag cgtcgattac aagatcttcg
     4741 aagatgtgcc ccagagcgcc aagatcaaca aggacgaccc cagcagaaac ttcagcgacg
     4801 ctctgaagaa gcagaggtac caggcaatcg tgtcacttta cctgaccgtg atgtacctca
     4861 tcacaaagaa cctggtgtac gtgaacagcc gttacgtgat cgcctttcac tgtctggaga
     4921 gggacgcctt cctgcacggc gtgaccctgc ccaagatgaa caagaagatc gtctacagcc
     4981 aactgaccac ccacctgctg accgacaaga actacaccac ctacggccac ctgaagaacc
     5041 agaagggcca caggaagtgg tacgtgcttg tgaagaacaa cctgcagaac agcgacatca
     5101 ccgccgtgtc cagcttcagg aacatcgtcg cccacatctc tgtggtgcgt aactccaacg
     5161 aatacatcag cggaatcgga gagctgcata gctacttcga gctgtaccac tacctggtgc
     5221 agagcatgat cgccaagaac aattggtacg ataccagcca tcagcccaag accgccgagt
     5281 acctgaacaa tctgaagaag caccacacgt actgcaagga tttcgtgaag gcctactgca
     5341 tccccttcgg ctacgtggtg cccaggtaca agaacctgac catcaacgag ctgttcgaca
     5401 ggaataaccc caaccccgag cccaaggagg aagttgcggc cgcactcgag caccaccacc
     5461 accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca
     5521 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
     5581 tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt
     5641 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     5701 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     5761 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     5821 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     5881 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     5941 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     6001 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     6061 aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     6121 tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc
     6181 gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa
     6241 aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc
     6301 ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc
     6361 gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa
     6421 tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc
     6481 atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg
     6541 aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag
     6601 gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg
     6661 gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat
     6721 aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc
     6781 atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc
     6841 gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca
     6901 tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt
     6961 ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag cagacagttt
     7021 tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     7081 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     7141 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     7201 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     7261 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     7321 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     7381 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     7441 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     7501 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     7561 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     7621 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     7681 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     7741 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     7801 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     7861 aggaagcgga ag
//
