LOCUS       sequence-245594-        9317 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses gRNA using fusion of R. toruloides 5S rRNA and tRNA-Arg as
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9317)
  AUTHORS   Schultz JC, Cao M, Zhao H
  TITLE     Development of a CRISPR/Cas9 system for high efficiency multiplexed 
            gene deletion in Rhodosporidium toruloides.
  JOURNAL   Biotechnol Bioeng. 2019 Apr 30. doi: 10.1002/bit.27001.
  PUBMED    31038202
REFERENCE   2  (bases 1 to 9317)
  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: "Biotechnol 
            Bioeng. 2019 Apr 30. doi: 10.1002/bit.27001."
COMMENT     Sequence Label: NM1-5S-tRNA-SgH
FEATURES             Location/Qualifiers
     source          1..9317
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1181..1810
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             2239..3312
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="MSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
                     QAAADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
                     KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
                     GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
                     ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
                     LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
                     LVMRYRNLIEGEASAGS"
     rep_origin      3378..3572
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(3808..3830)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3916..4056
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3971..3990
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4071..4088)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4242..4830)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4322..4341)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             4903..5718
                     /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             4903..5718
                     /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     complement(4976..4995)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     misc_feature    5954..5978
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     misc_RNA        6262..6337
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             8808..8825
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      8829..9081
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     misc_feature    9180..9204
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
ORIGIN
        1 agtactttga tccaacccct ccgctgctat agtgcagtcg gcttctgacg ttcagtgcag
       61 cctgaaaacg acatgtcgca caagtcctaa gttacgcgac aggctgccgc cctgcccttt
      121 tcctggcgtt ttcttgtcgc gtgttttagt cgcataaagt agaatacttg cgactagaac
      181 cggagacatt acgccatgaa caagagcgcc gccgctggcc tgctgggcta tgcccgcgtc
      241 agcaccgacg accaggactt gaccaaccaa cgggccgaac tgcacgcggc cggctgcacc
      301 aagctgtttt ccgagaagat caccggcacc aggcgcgacc gcccggagct ggccaggatg
      361 cttgaccacc tacgccctgg cgacgttgtg acagtgacca ggctagaccg cctggcccgc
      421 agcacccgcg acctactgga cattgccgag cgcatccagg aggccggcgc gggcctgcgt
      481 agcctggcag agccgtgggc cgacaccacc acgccggccg gccgcatggt gttgaccgtg
      541 ttcgccggca ttgccgagtt cgagcgttcc ctaatcatcg accgcacccg gagcgggcgc
      601 gaggccgcca aggcccgagg cgtgaagttt ggcccccgcc ctaccctcac cccggcacag
      661 atcgcgcacg cccgcgagct gatcgaccag gaaggccgca ccgtgaaaga ggcggctgca
      721 ctgcttggcg tgcatcgctc gaccctgtac cgcgcacttg agcgcagcga ggaagtgacg
      781 cccaccgagg ccaggcggcg cggtgccttc cgtgaggacg cattgaccga ggccgacgcc
      841 ctggcggccg ccgagaatga acgccaagag gaacaagcat gaaaccgcac caggacggcc
      901 aggacgaacc gtttttcatt accgaagaga tcgaggcgga gatgatcgcg gccgggtacg
      961 tgttcgagcc gcccgcgcac gtctcaaccg tgcggctgca tgaaatcctg gccggtttgt
     1021 ctgatgccaa gctggcggcc tggccggcca gcttggccgc tgaagaaacc gagcgccgcc
     1081 gtctaaaaag gtgatgtgta tttgagtaaa acagcttgcg tcatgcggtc gctgcgtata
     1141 tgatgcgatg agtaaataaa caaatacgca aggggaacgc atgaaggtta tcgctgtact
     1201 taaccagaaa ggcgggtcag gcaagacgac catcgcaacc catctagccc gcgccctgca
     1261 actcgccggg gccgatgttc tgttagtcga ttccgatccc cagggcagtg cccgcgattg
     1321 ggcggccgtg cgggaagatc aaccgctaac cgttgtcggc atcgaccgcc cgacgattga
     1381 ccgcgacgtg aaggccatcg gccggcgcga cttcgtagtg atcgacggag cgccccaggc
     1441 ggcggacttg gctgtgtccg cgatcaaggc agccgacttc gtgctgattc cggtgcagcc
     1501 aagcccttac gacatatggg ccaccgccga cctggtggag ctggttaagc agcgcattga
     1561 ggtcacggat ggaaggctac aagcggcctt tgtcgtgtcg cgggcgatca aaggcacgcg
     1621 catcggcggt gaggttgccg aggcgctggc cgggtacgag ctgcccattc ttgagtcccg
     1681 tatcacgcag cgcgtgagct acccaggcac tgccgccgcc ggcacaaccg ttcttgaatc
     1741 agaacccgag ggcgacgctg cccgcgaggt ccaggcgctg gccgctgaaa ttaaatcaaa
     1801 actcatttga gttaatgagg taaagagaaa atgagcaaaa gcacaaacac gctaagtgcc
     1861 ggccgtccga gcgcacgcag cagcaaggct gcaacgttgg ccagcctggc agacacgcca
     1921 gccatgaagc gggtcaactt tcagttgccg gcggaggatc acaccaagct gaagatgtac
     1981 gcggtacgcc aaggcaagac cattaccgag ctgctatctg aatacatcgc gcagctacca
     2041 gagtaaatga gcaaatgaat aaatgagtag atgaatttta gcggctaaag gaggcggcat
     2101 ggaaaatcaa gaacaaccag gcaccgacgc cgtggaatgc cccatgtgtg gaggaacggg
     2161 cggttggcca ggcgtaagcg gctgggttgt ctgccggccc tgcaatggca ctggaacccc
     2221 caagcccgag gaatcggcgt gagcggtcgc aaaccatccg gcccggtaca aatcggcgcg
     2281 gcgctgggtg atgacctggt ggagaagttg aaggccgcgc aggccgccca gcggcaacgc
     2341 atcgaggcag aagcacgccc cggtgaatcg tggcaagcgg ccgctgatcg aatccgcaaa
     2401 gaatcccggc aaccgccggc agccggtgcg ccgtcgatta ggaagccgcc caagggcgac
     2461 gagcaaccag attttttcgt tccgatgctc tatgacgtgg gcacccgcga tagtcgcagc
     2521 atcatggacg tggccgtttt ccgtctgtcg aagcgtgacc gacgagctgg cgaggtgatc
     2581 cgctacgagc ttccagacgg gcacgtagag gtttccgcag ggccggccgg catggccagt
     2641 gtgtgggatt acgacctggt actgatggcg gtttcccatc taaccgaatc catgaaccga
     2701 taccgggaag ggaagggaga caagcccggc cgcgtgttcc gtccacacgt tgcggacgta
     2761 ctcaagttct gccggcgagc cgatggcgga aagcagaaag acgacctggt agaaacctgc
     2821 attcggttaa acaccacgca cgttgccatg cagcgtacga agaaggccaa gaacggccgc
     2881 ctggtgacgg tatccgaggg tgaagccttg attagccgct acaagatcgt aaagagcgaa
     2941 accgggcggc cggagtacat cgagatcgag ctagctgatt ggatgtaccg cgagatcaca
     3001 gaaggcaaga acccggacgt gctgacggtt caccccgatt actttttgat cgatcccggc
     3061 atcggccgtt ttctctaccg cctggcacgc cgcgccgcag gcaaggcaga agccagatgg
     3121 ttgttcaaga cgatctacga acgcagtggc agcgccggag agttcaagaa gttctgtttc
     3181 accgtgcgca agctgatcgg gtcaaatgac ctgccggagt acgatttgaa ggaggaggcg
     3241 gggcaggctg gcccgatcct agtcatgcgc taccgcaacc tgatcgaggg cgaagcatcc
     3301 gccggttcct aatgtacgga gcagatgcta gggcaaattg ccctagcagg ggaaaaaggt
     3361 cgaaaaggtc tctttcctgt ggatagcacg tacattggga acccaaagcc gtacattggg
     3421 aaccggaacc cgtacattgg gaacccaaag ccgtacattg ggaaccggtc acacatgtaa
     3481 gtgactgata taaaagagaa aaaaggcgat ttttccgcct aaaactcttt aaaacttatt
     3541 aaaactctta aaacccgcct ggcctgtgca taactgtctg gccagcgcac agccgaagag
     3601 ctgcaaaaag cgcctaccct tcggtcgctg cgctccctac gccccgccgc ttcgcgtcgg
     3661 cctatcgcgg ccgctggccg ctcaaaaatg gctggcctac ggccaggcaa tctaccaggg
     3721 cgcggacaag ccgcgccgtc gccactcgac cgccggcgcc cacatcaagg caccctgcct
     3781 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     3841 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     3901 tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg
     3961 cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata
     4021 ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact
     4081 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     4141 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     4201 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     4261 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     4321 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     4381 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     4441 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     4501 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     4561 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     4621 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     4681 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     4741 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     4801 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     4861 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagcca tattcaacgg
     4921 gaaacgtctt gctctaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat
     4981 aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag
     5041 cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca
     5101 gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat
     5161 tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg gaaaacagca
     5221 ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg
     5281 ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta
     5341 tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt
     5401 gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca taaacttttg
     5461 ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt
     5521 gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac
     5581 caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg
     5641 ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg
     5701 ctcgatgagt ttttctaatc acaggcagca acgctctgtc atcgttacaa tcaacatgct
     5761 accctccgcg agatcatccg tgtttcaaac ccggcagctt agttgccgtt cttccgaata
     5821 gcatcggtaa catgagcaaa gtctgccgcc ttacaacggc tctcccgctg acgccgtccc
     5881 ggactgatgg gctgcctgta tcgagtggtg attttgtgcc gagctgccgg tcggggagct
     5941 gttggctggc tggtggcagg atatattgtg gtgtaaacaa attgacgctt agacaactta
     6001 ataacacatt gcggacgttt ttaatgtact gaattaacgc cgaattgaat tcatctgcgg
     6061 ccataccgcg atgaacacac cgcgtctcgt ccgatccgcg aagttaagca tcgcaggggc
     6121 cagagagtat tgccgtgggt gaccaggcga gaacactgtg ctgccgcagg tggcggtgtg
     6181 gccaagtggc acggcacctg tttccggcgc aggagatcga gggttcgatc cccttcaccg
     6241 tcgaggtctt caggaagacg agttttagag ctagaaatag caagttaaaa taaggctagt
     6301 ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttttttggta cccgggctgc
     6361 agaactacgc cctctcacac ccaacttccg actcgaccgg cggtacgagc acgacctact
     6421 tctactgcct gccatcgaca tccgggcggg tcgctgccta ccctgtgcgt tctgcgccct
     6481 ccctcgtctc gggaggcagt gtctgacaga agctttgcgc gcagtacccc gtcaagatgc
     6541 aactctacgc aacgttcggc acagaagtcg ccaagctccg cgcatcgccg cctcaagctc
     6601 tcgcgctgcc cgacggtgtc gtctattacg aggcggagaa gctcgagttg ccggctttgc
     6661 cagcggcggt caaggttgag gtggagacgg agaaggcggg agtagcgggg gaggacaatg
     6721 aggcgaaggg tgagatggtg ctggtggaga ctcttacggt ggagcaggag gagattgaat
     6781 tgggctcggg agtcgtgcag attgaggagt cgttgctcgt caagctggag gtcagcggct
     6841 gatccttccg ttcgttgcaa ggatcgtctg catgttcccg ggtcgcttct ctcaatgaca
     6901 caacctggag agcgctcccg tcagcgagaa tcgaggacat tccgcagctc gtgagcaagc
     6961 ggaggtgcga ggctccctcg aaagctgcgc ctcttcagac ggcttgttct ctcctgctct
     7021 ggtgggctgg cctgacatgt aatgtgctcc gccgcaagtc cgtcgtcggt ctcaattcga
     7081 cgttgaaagg gcatagcgca aggaagaacc ctctgcggac atgcagaatt actggctcgc
     7141 ctgctccttc gtctactgga taagtcctgt ctcgttaaag ccccaacgtc gtttttcgac
     7201 gtttgtaagg cgcaagaggt gctatgggct acgcaggaag ctgagaggac atagaagtcg
     7261 ggggaggaac ggcgcagagc ggcagttgcg gaagcatgag gaaagcgaga cggtccagca
     7321 tctgcagcgc caatccgcaa tctcctggtt gagcctgcac cggaagcgtc ggaacagtat
     7381 gcgcagagtc gaacgcaagt aagaaagacg caccctcaca ctcgcttact tcgagccata
     7441 caacggatca aagctgcgcg tatctcggct tgtaagggcc ggaaagcaac ctcggagatg
     7501 gacacgtcac atcaccaact tatcgatctc ggccgtcgac gtcgcagaga gggcgagaga
     7561 agcggtgaag gagggaaaca acccctcgag agcatgatcc gaccgaatct gcagcgcagg
     7621 aagccgttac aagcccgcct cgagcgcagg tcgggtccag ccgggggacg aaacgcgcga
     7681 ggctgattcg tgagcgaagg aagccgcatc gacaagttcg ctcccctttg ccctctttcc
     7741 catcacccgt tctcgcctta cccgctcaga acaacaccag atcactcaca atgccggagc
     7801 tcatggcgac gtcggtcgag aagttcctca tcgaaaagtt cgactcggtc tcggacctca
     7861 tgcaactctc ggagggagag gaatcgcgcg cgttctcgtt cgacgtcgga ggccgcggat
     7921 acgtcctccg cgtcaactcg tgtgcggacg gattctacaa ggatcggtac gtctaccgcc
     7981 attttgcgtc ggcggcgctc ccgatccccg aagtcctcga catcggagag ttctcggaat
     8041 ccctcacgta ctgtatctcg cgccgggcgc aaggagtcac gctccaagat ctcccggaga
     8101 cggaactccc ggcggtcctc caaccggtcg cggaagcgat ggacgcgatc gcggccgcgg
     8161 acctctcgca aacgtcggga ttcggaccgt ttggaccgca aggaatcgga caatacacga
     8221 cgtggcgcga cttcatctgt gcgatcgcgg atccccatgt ctaccactgg caaacggtca
     8281 tggatgacac ggtctcggcg tcggtcgcgc aagcgctcga cgagctcatg ctctgggcgg
     8341 aggactgtcc ggaggtccgc cacctcgtcc acgcggactt tggatcgaac aacgtcctca
     8401 cggacaacgg acgcatcacg gcggtcatcg actggtcgga ggcgatgttt ggagactcgc
     8461 aatacgaggt cgcgaacatc ttcttctggc gcccgtggct cgcgtgcatg gagcaacaga
     8521 cgcgctactt cgagcgccgc cacccggagc tcgcgggatc gccgcgcctc cgcgcgtaca
     8581 tgctccgcat cggcctcgac caactctacc agtcgctcgt cgacggaaac ttcgacgatg
     8641 ccgcgtgggc ccaaggacgc tgcgacgcga ttgtccgctc gggagcggga accgtgggac
     8701 gcacgcaaat tgcgcggcgc tcggcggccg tctggacgga tggatgtgtc gaagtcctcg
     8761 cggattcggg aaaccggcgc ccgtcgacgc gcccgcgggc gaaagaacac caccatcacc
     8821 atcactagga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg
     8881 gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca
     8941 tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca
     9001 tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg
     9061 tgtcatctat gttactagat cgggcctgga tcctctagag tcgacctgca ggcatgcaag
     9121 cttagcttga gcttggatca gattgtcgtt tcccgccttc agtttaaact atcagtgttt
     9181 gacaggatat attggcgggt aaacctaaga gaaaagagcg tttattagaa taacggatat
     9241 ttaaaagggc gtgaaaaggt ttatccgttc gtccatttgt atgtgcatgc caaccacagg
     9301 gttcccctcg ggatcaa
//
