LOCUS       sequence-481113-        7932 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 7932)
  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 7932)
  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_ancCas9d_Plasmid
FEATURES             Location/Qualifiers
     source          1..7932
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             292..312
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2602..2619
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2672..2690)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2686..2733
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2770..3225
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2857..2876)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3067..3088
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3317..4132)
                     /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(3317..4132)
                     /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     4040..4059
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4124..4143
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4254..4842
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4743..4762
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4996..5013
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5028..5167
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5093..5112)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5253..5275
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5269..5460)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6269..7351)
                     /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     7313..7332
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7352..7429)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7635..7654
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     7738..7757
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7738..7756
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7757..7781
                     /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             7796..7818
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             7837..7854
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             7864..7881
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             7885..7917
                     /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"
ORIGIN
        1 atgagcgatt ccgaagttaa tcaagaagcg aaacctgagg tgaaaccgga ggtgaaaccg
       61 gaaacacata ttaatttaaa agtctctgat gggagctctg aaattttttt taaaatcaaa
      121 aaaacaacac ctcttcggag attgatggaa gcctttgcaa aacgccaagg caaagaaatg
      181 gattcactgc gctttctgta cgacggtatt cgtattcagg cggaccagac accagaggat
      241 ctggacatgg aagataatga tatcattgaa gcccatcgcg aacaaattgg ggagaactta
      301 tattttcagg gtcacatgat ggcagcggct ttctcaatgg agaaggagct ggtgttaggt
      361 attgactatg gcgggaagta cactggtctg gcagtagtgg atagacgcaa caatcaagta
      421 ctgtatgccc ggacggttaa gatgcgagac gacgttgccg acatacttaa aggtcgtcgc
      481 gagcaaagag gtattagacg tacccaacag acaaagaaga agcgtctccg cgagttgaag
      541 aagtacttaa actcgatcgg actgtatgac gagtccacgg aaacatttaa gaccatctac
      601 agtctcgcgc acaaaagagg ctacgactat gcggacatgc cagaggagaa gaccagcgaa
      661 gagatagaag agatggacga ggaagaacgc aagcaatggg aaaaggagaa gaaagagtgg
      721 gaagagacgc aacgtaacag tagacatcgt gaagaggtcc tgaaggatgt tcgaaaagtt
      781 atgagtgagg ggaacgcaag tgaggagcaa attaaacggg tagagtccat cttcaacaaa
      841 cagtatcgcc ctaagcgttt caataaccgt atcctcacga agtgcaaagt ttgtgggaag
      901 aacacccctc tccgcagaaa tgttcgagag ttgttactgg aaaatatagt gagattctta
      961 cctctggagt ctgaagagaa ggagacgctg aagcaaacca tactggagaa aggccaacag
     1021 gagaatatca acaagatatt tcgcaagttg aagttcaacc agaaggattg gccaggaaag
     1081 aaccttacag acattgcaaa gaataagtta cctggacgtc tccctttctg taaagagcat
     1141 ttcgcagaga acgagaagtt cacaactata gagaaatcta ctttccgcct ggcaccgtcg
     1201 ctgaagacta agattgaaaa tgtgcttaca gtgataaagg acgaaatcct gccaaatttc
     1261 actcttaaca aagtggtgat ggaaagcaac aacttcgaca tcgcagccaa aacgaagggc
     1321 aagaagcgtc ttgcaaagga agagtattca aagggacacc gcgagaacaa ggagacttta
     1381 aaggaagcat tactgagaga aacggacggc cgctgtgtgt actgtggaaa atcaattgac
     1441 ctgtctaacg cgaacaaaga ccacatatac cctagaaagg ccggcggtct taacattttc
     1501 gctaatctcg ttgcatgctg ccgcagttgt aatgagaaca agaaggggcg tacaccgctt
     1561 gaaagcggga taatgcccaa gcctgaagtg gtagccacta tcaagaatga cctgaagaag
     1621 aagatattgg aagatgcgca acaaattaac accctggact tcaataagta tatgagtcat
     1681 gcgtcgattg gttggcgcca tatgcgcgat cggcttcgag aactgaccgg aaataagaag
     1741 ctgccaatcg agcgccaatc aggaatagta actgcatact tccgccgctg gtggggtttc
     1801 aagaaagagc gtgggaatga caaacaccat gcgttggacg cagttattct tgcctcgcgt
     1861 aaggactact cagacgacgg ccttgttgat atgacactta agcctgcaaa caacgacggc
     1921 cgtgagttcg acccagagaa gcacatcaaa gagatgaagg agttcaagcg cgacaagggt
     1981 tccaagggat ccgcgctgca cgatcgaaac cccttatcat acaagaatgg aataatcacg
     2041 cggcgcttca tggtaactga gattgagcgc ggcaaggaag acgcggtcat atcagaagag
     2101 taccgtgaaa agcttaagga agcattcgat cggttcggta taaagaatgg gaagtgtctt
     2161 acggacaagc aggcgaaaga agcaggcctt tacatcaaga agaacgaggg tgttatgagc
     2221 cttaagtgca gtgttaaagg caccggtcca ggacaaatga taagaatcaa gaataacgtt
     2281 ttcaaaacta atgtacacaa tgtcggagta gcagtatact tggacgaaaa gggtaagaag
     2341 cgtgcatgtg aacttaagaa tccacgtctt tccaagcatt tcgtggaacc tcagcctcag
     2401 gtcaaaggca aaatcctctt caccttacgt cgtggcgaca tggtaactgt ggaaggtgga
     2461 gatgccatat acagaatcaa gaagttgggc acatccccag ttatcgagat cgttccaggg
     2521 tcggacggaa agactaagac agtatctgca acaaaattga ccaaggctaa caacgcgaag
     2581 taataagcgg ccgcactcga gcaccaccac caccaccact gagatccggc tgctaacaaa
     2641 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     2701 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg
     2761 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     2821 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     2881 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     2941 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     3001 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     3061 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     3121 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     3181 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
     3241 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     3301 gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca
     3361 tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact
     3421 caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc
     3481 caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat
     3541 caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga
     3601 cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt
     3661 tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat
     3721 tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt
     3781 cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg
     3841 tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa
     3901 attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt
     3961 tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg
     4021 cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt
     4081 tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc
     4141 ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt
     4201 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     4261 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     4321 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     4381 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     4441 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     4501 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     4561 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     4621 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     4681 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     4741 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     4801 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     4861 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     4921 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     4981 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     5041 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
     5101 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     5161 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     5221 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     5281 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     5341 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     5401 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     5461 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     5521 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     5581 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     5641 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     5701 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     5761 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     5821 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     5881 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc
     5941 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
     6001 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
     6061 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
     6121 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
     6181 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     6241 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     6301 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     6361 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     6421 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     6481 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     6541 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     6601 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     6661 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     6721 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     6781 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     6841 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     6901 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     6961 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     7021 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     7081 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     7141 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     7201 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     7261 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     7321 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     7381 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     7441 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     7501 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     7561 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
     7621 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
     7681 gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa
     7741 tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt
     7801 ttaactttaa gaaggagata taccatgggc agcagccatc atcatcatca tcacagcagc
     7861 ggcctggtgc cgcgcggcag ccatatggct agcatgactg gtggacagca aatgggtcgc
     7921 ggatccgaat tc
//
