LOCUS       sequence-208499-        6568 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses E. coli codon optimized RspWYL1 in the pET28a backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6568)
  AUTHORS   Yan WX, Chong S, Zhang H, Makarova KS, Koonin EV, Cheng DR, Scott DA
  TITLE     Cas13d Is a Compact RNA-Targeting Type VI CRISPR Effector Positively
            Modulated by a WYL-Domain-Containing Accessory Protein.
  JOURNAL   Mol Cell. 2018 Mar 9. pii: S1097-2765(18)30173-4. doi: 
            10.1016/j.molcel.2018.02.028.
  PUBMED    29551514
REFERENCE   2  (bases 1 to 6568)
  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: "Mol Cell. 
            2018 Mar 9. pii: S1097-2765(18)30173-4. doi: 
            10.1016/j.molcel.2018.02.028."
COMMENT     Sequence Label: pET28a-MH6-RspWYL1
FEATURES             Location/Qualifiers
     source          1..6568
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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)."
     promoter        join(53..58,59..76,77..82)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     RBS             108..130
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             162..179
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        1374..1408
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     primer_bind     complement(1503..1521)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1517..1564
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1601..2056
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1688..1707)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1898..1919
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2148..2963)
                     /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(2148..2963)
                     /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     2871..2890
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2955..2974
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3085..3673
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3574..3593
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3827..3844
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3859..3998
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3924..3943)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4084..4106
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4100..4291)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5100..6182)
                     /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     6144..6163
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6183..6260)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6466..6485
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gatttacact
       61 ttatgcttcc ggctcgtatg tttctagaga attcggatcc aaataatttt gtttaacttt
      121 aagaaggaga tttaaatatg aaaatcgaag aaggtaaagg tcaccatcac catcaccaca
      181 tgctgattcc gcctagcacc tttctgccga aacgtgataa aaatgttccg tatattgcag
      241 aggttcagag cattccgctg agcccgagcg catatagcgt tattatcaaa gataagagca
      301 tctttgaaac cagcctgagt ccgaatggta gcgttagcat gagcagcttt ctgaccagca
      361 tttttgatag cgcctatatc gcaagcctga aatacaaaag cgacgacaac tataaataca
      421 ttggtattcc gctgctgaac gcctttgtta aatggcagat tgaagaaatt gatgatggcc
      481 tggatgacaa gagcaaagaa atcattaaaa gctatctgat cagcaagctg agcgccaaat
      541 atgaaaaaac caaaaccgaa aatgccgttc gtgttcgtct gagcatttgt cgtgatctgt
      601 atgataccct gagcagcgac gatctgtatt atgaaaataa agtgtatagc agcaccctgc
      661 gtcgttttct gaaagcagtt tatgaagatt atgcactgct gagcgattgt gaacgtgaac
      721 gtctgatttt tgccgataac atcatcaaaa tcaacgaagt gattaagcag aatggcagcc
      781 gttattatag cttcatttat gcctatagca acatgtacag ccgtgaaaaa cgtcgtattc
      841 gcctgattcc gtatcgtatt gtttccgatg agtacaaaat gtataactac ctggtttgtc
      901 tgagcgacga aaaaagcgca ggtaaagaat ttaaagcaga tagctgtcgt attagccgtc
      961 tgagcggtct gagtattgca gaaaaactga gccagaaaga atatagcagc gtgaccgaat
     1021 atgaacgcct gaaagaagtt cacgttaaaa gcgttaaaca cctgttaagc gatccgcgtt
     1081 ttggtagtga tgaaagcgat attagcaaag tgtatctgac cgaaaaaggc gtggaaatgt
     1141 ttggcaaaat tctgtatcag cgtccgattc tgaaaggcaa tgagaaaccg aaacctaatg
     1201 ccgtgaatga atttatcagc cctccgattc aggtgaaata ctactttaac aaattcggca
     1261 aagacggcgt tattctgagc ccgtctgata gctttgaaga gatgcgtacc ctgtatgttg
     1321 aaggtgccga agcatataat cgtgaagttg aaatgtaata aggatccctc gagttgacag
     1381 ctagctcagt cctaggtata atgctagcag agaccatggc caccggtctc tgctaaagat
     1441 atgaggagct caaactcgag ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
     1501 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     1561 tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca
     1621 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     1681 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     1741 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     1801 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     1861 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     1921 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     1981 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     2041 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     2101 tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca
     2161 tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga
     2221 aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga
     2281 tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc
     2341 tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag
     2401 aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg
     2461 tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga
     2521 cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc
     2581 aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc
     2641 tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg
     2701 ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc
     2761 tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca
     2821 tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc
     2881 catttatacc catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac
     2941 gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt
     3001 tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     3061 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     3121 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     3181 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     3241 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     3301 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     3361 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     3421 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     3481 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     3541 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     3601 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     3661 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     3721 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     3781 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     3841 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     3901 acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     3961 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
     4021 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     4081 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
     4141 tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg
     4201 cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca
     4261 tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt
     4321 tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg
     4381 atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc
     4441 gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg
     4501 ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg
     4561 ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg
     4621 ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt
     4681 cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca
     4741 cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac
     4801 gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg
     4861 caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc
     4921 agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg
     4981 cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
     5041 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
     5101 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     5161 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
     5221 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
     5281 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
     5341 catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc
     5401 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
     5461 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
     5521 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc
     5581 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
     5641 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
     5701 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
     5761 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
     5821 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
     5881 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
     5941 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
     6001 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
     6061 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
     6121 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
     6181 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
     6241 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag
     6301 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
     6361 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
     6421 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
     6481 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
     6541 gaggatcgag atctcgatcc cgcgaaat
//
