LOCUS       sequence-481114-        7929 bp    DNA     circular SYN 11-AUG-2025
DEFINITION  Expression plasmid codon-optimized for E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7929)
  AUTHORS   Ocampo RF, Bravo JPK, Dangerfield TL, Nocedal I, Jirde SA, Alexander
            LM, Thomas NC, Das A, Nielson S, Johnson KA, Brown CT, Butterfield 
            CN, Goltsman DSA, Taylor DW
  TITLE     DNA targeting by compact Cas9d and its resurrected ancestor.
  JOURNAL   Nat Commun. 2025 Jan 7;16(1):457. doi: 10.1038/s41467-024-55573-4.
  PUBMED    39774105
REFERENCE   2  (bases 1 to 7929)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 11, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2025-01-7"; volume: "16(1):457. doi"; pages: " 
            10.1038/s41467-024-55573-4"
COMMENT     Sequence Label: pET28_Cas9d_Plasmid
FEATURES             Location/Qualifiers
     source          1..7929
                     /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)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..144
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             148..180
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             487..507
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             976..987
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             2794..2811
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2864..2882)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2878..2925
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2962..3417
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3049..3068)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3259..3280
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3509..4324)
                     /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(3509..4324)
                     /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     4232..4251
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4316..4335
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4446..5034
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4935..4954
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5188..5205
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5220..5359
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5285..5304)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5445..5467
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5461..5652)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6461..7543)
                     /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     7505..7524
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7544..7621)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7827..7846
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcctgg tgccgcgcgg cagccatatg gctagcatga ctggtggaca gcaaatgggt
      181 cgcggatccg aattcatgag cgattccgaa gttaatcaag aagcgaaacc tgaggtgaaa
      241 ccggaggtga aaccggaaac acatattaat ttaaaagtct ctgatgggag ctctgaaatt
      301 ttttttaaaa tcaaaaaaac aacacctctt cggagattga tggaagcctt tgcaaaacgc
      361 caaggcaaag aaatggattc actgcgcttt ctgtacgacg gtattcgtat tcaggcggac
      421 cagacaccag aggatctgga catggaagat aatgatatca ttgaagccca tcgcgaacaa
      481 attggggaga acttatattt tcagggtcac atgatggcag cggctttctc aatggagaga
      541 gaactggtct tgggtatcga ctacggtgga aagtatacag gattagcagt ggttgataga
      601 cgtcataatc aggttctgta tgcaaaccgc ctcaaaatga gagatgatgt cgctggtatc
      661 ctgaaagacc gccggaagca acgagggatt cgccgtacag cgcagacaaa aaaaaaacgc
      721 ctgcgggaac tgaagaatta tttgaaaagt ataggttaca acgaaagtac cgcaaccttt
      781 gagacggtct atagcttagc ccataaacga ggatacgatt atgcggatat gcctgaagaa
      841 aagacctcgg aggaaataga agcaatggac gtggaagagc gtaagcagtg ggaaaaagaa
      901 aagcaggaat gggaagaaac aaagcgcaat tcccgccatc ggaaagaagt ggtgaaagat
      961 gtgcataagg caatgataga aggccgtgca actgaggaac agattaagag agtggaacgc
     1021 atattcaaca aacagtacag acccaaacgt ttcaataatc gcatacttac caaatgcaaa
     1081 gtggaagatt gtggggtcaa tacacctctg cgtaagaacg tgcgtgatct gttaatcgaa
     1141 aatattgtgc gtttcttccc gattgaacag tcggaaaagg acaatctgaa agacgcagtc
     1201 ctggataaaa atcgccggga ggaagtgaaa agtttttttc gcaaacataa aactgacgaa
     1261 cacattcgca aacaagtgta tgatatcgcg gacaataaat tgagtgggcg taccgttttc
     1321 tgcaaggaac atattctcga gcatacagaa cactcgaagg aagaaagaaa agtatttcgg
     1381 ctcgcccctt ctctcaaaac taaaattgaa aatgttttag cagtaattaa agatgagata
     1441 ttaccaaaat tcacggtaaa caaggttgtt atggagtcga acaactttga tattgcagcc
     1501 aaaacccagg gcaagaaaag attagcaaaa gaagagtatg gcaaaggtcc tcgcgaaggc
     1561 aaggagaccc gcaaggaagc actgcttcgt gaaacagatg ggcgttgtat ttattgcgga
     1621 aagagtatcg atatttctaa tgcccacgat gaccatattt ttcctagaaa agcaggaggg
     1681 ctgaacatct ttgcgaacct ggtggcttgc tgcgcggtct gtaatgaaaa taaaaagggg
     1741 cgcacgccac tggaatcggg catctctcct aaacctgaaa ttattgcgtt tatgaagaat
     1801 gatcttaaaa aaaaaattct ggaggacgcg cggaatataa atacggtgga ctttaataaa
     1861 tacatgtctc acgcatcaat cgggtggcgc tatatgcggg atcgtcttcg tgaatcagca
     1921 ggtaataaga aactcccgat agaacgccag tcgggtatct atacggccta ttttcgtaga
     1981 tggtgggggt tcaaaaaaga acgtggcaac acgctgcacc acgcacttga cgcagtgatt
     2041 ttagcatcac ggaaaggcta ttccgatgat ggtttagtgg atatgacgct taaacctaag
     2101 tataacaaag gtggagaatt tgatccggaa aagcacttac ccgagcctat cgagtttaag
     2161 atggataagg gtagtagagg ttctgccttg cacgatcgta atccattatc gtataaaaaa
     2221 ggcatcatta cacggcgttt catggttact gaaatcgagt gtggaaaaga agacgatgtg
     2281 atttccgaaa cttatcggga aaaactgaag gaagcattta agcgttttga tacgaagaaa
     2341 ggtaaatgcc tgaccgataa agaggctaaa gaagctggat tctgtattaa aaaaaatgaa
     2401 cttgtgatga gcctgaaatg cagcattaag ggtactgggc cgggacagat gatacgtatt
     2461 aataacaatg tgttcaagac taatgtacat aacgtgggcg tggacgtcta tctggatgaa
     2521 aaaggcaaaa aaaaagctta cgagcgtaaa aatccccggc tgtcaaaaca ctttatagaa
     2581 cctccgcctc agccaaatgg gcgggtgtcc ttcaccctga aacggcgtga tatggtgacc
     2641 gtagaagggg aagacgctat ataccggatc aagaagcttg gtacctctcc taccatcgag
     2701 gcagtggttg gctctgatgg caaaacacgc acggtttcgg cgacaaaatt aaccaaagcg
     2761 aattccgcag agtaataagc ggccgcactc gagcaccacc accaccacca ctgagatccg
     2821 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta
     2881 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
     2941 atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     3001 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     3061 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     3121 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     3181 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     3241 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     3301 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     3361 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     3421 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     3481 aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact
     3541 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
     3601 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
     3661 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
     3721 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca
     3781 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
     3841 caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt
     3901 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
     3961 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg
     4021 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     4081 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
     4141 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
     4201 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
     4261 cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc
     4321 tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa
     4381 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     4441 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     4501 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     4561 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     4621 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     4681 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     4741 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     4801 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     4861 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     4921 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     4981 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     5041 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     5101 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     5161 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     5221 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc
     5281 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     5341 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     5401 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     5461 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
     5521 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
     5581 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
     5641 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
     5701 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
     5761 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
     5821 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
     5881 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
     5941 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
     6001 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
     6061 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg
     6121 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt
     6181 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat
     6241 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg
     6301 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
     6361 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg
     6421 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
     6481 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     6541 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
     6601 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
     6661 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
     6721 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
     6781 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
     6841 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
     6901 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
     6961 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
     7021 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
     7081 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
     7141 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
     7201 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
     7261 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
     7321 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
     7381 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
     7441 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
     7501 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     7561 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     7621 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     7681 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     7741 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     7801 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     7861 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
     7921 ccgcgaaat
//
