LOCUS       sequence-454353-        6368 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Triple sgRNAs expression plasmid in Kluyveromyces marxianus..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6368)
  TITLE     Wang lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6368)
  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: 
            "Unpublished"
COMMENT     Sequence Label: LHZ1494
FEATURES             Location/Qualifiers
     source          1..6368
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     485..504
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(723..827)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     895..913
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(951..973)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1073..1092
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1173..1191
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1173..1190
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_RNA        2619..2694
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     terminator      2695..2714
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     promoter        complement(2737..2755)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        2769..3037
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        3059..3134
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     terminator      3135..3154
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     gene            3233..4808
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        3233..3576
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             3577..4605
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             3577..4605
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     terminator      4611..4808
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     misc_RNA        5040..5115
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     terminator      5116..5135
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     promoter        complement(5195..5213)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5300..5317)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5471..6059)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5551..5570)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6230..6368,1..653,654..722))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
       61 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
      121 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
      181 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
      241 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
      301 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
      361 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
      421 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
      481 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
      541 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
      601 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
      661 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
      721 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
      781 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
      841 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
      901 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
      961 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     1021 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca
     1081 gagcagattg tactgagagt gcaccatatg gacatattgt cgttagaacg cggctacaat
     1141 taatacataa ccttatgtat catacacata cgatttaggt gacactatag aacgcggccg
     1201 ccagctgaag ctttcgattg aagttttgtc caactatcca ctatggatat gcgttttgtt
     1261 gattaacctt aaataacacg tatttcgcat tttccaaaag ccttttttca taactacaaa
     1321 ctaactattt ttgtttattt tacatcagta aattatgcgc agaaaatatg taaggctata
     1381 tactcaatat agtggaagag cctctggcta cttaatctgg gttcatatat tctgtcagtg
     1441 gtatagtaaa tttaaatagt gaatttgggc gcatgtagag agatcttttg attaataacc
     1501 cagtatttga ttcttgaatg ttatttgtct cttattaagt atttcagttt gatttatata
     1561 ttttgaaagt aattgttgct ttaccttcca aacaataaaa aaatataaaa aaatgtaaaa
     1621 aatataataa atattaaata aaatactact tgtttgaaaa tcagagaaaa tccaccaaaa
     1681 tatcaatcat ttcaaggatt tccgaaccaa gttcgagata gtcctttaag gtcagtaaaa
     1741 ttcagttgca cgtataacag atatttttca ttttgttcca atttaaaagt cccccatttt
     1801 taaaattatt gaaaataaaa attaaaaaat taaaaggaat ctctctatgt cactttaaaa
     1861 taaataaatt gaaaatgata tatcgttaaa agtcaaccgc aacaccacct atttctaaga
     1921 ggagagttct aaaaaaatca tagtaccaca caggtaaact aaaacagcta aattcaacat
     1981 aatacctgtg gatataatta catacaaaaa tataattaaa aaaatacatt aaaaataatt
     2041 tatttttgta aaacccataa aatatatttt actttcggaa caacttttta acttataatt
     2101 ttgttttaaa taaaaacgtt gtatttaaaa ataataaaat attaagtaaa aatttaaagc
     2161 atatttattt aaaatataaa atactactaa aattactcta aacttcaaaa taaaaaaata
     2221 ataaattata tagttttaaa attaataatt tgtatacacg tgaccagacc ataatagttt
     2281 tcttttcttg aaactgccat gaatttaata gatttttttt acacatattt acagtaagtt
     2341 ttgtttttac gttgaattaa tattgtatac gtacttagaa ggataacttc caactatgaa
     2401 tatgtaggtt aaaaggtaaa tagagaagcc ttacttttat cagaaatgaa aggagctcgt
     2461 cgacaatttc tcttctacca cgaactcgac actgtagtct agtggtttag gatactcccc
     2521 ttccacttaa caagttgctt cctaaaaaga agtaacaagg cgagggagtg acacgggttc
     2581 gaatcccgtc agtgtcaaat tgggggaatt ataagcgtgt tttagtactc tggaaacaga
     2641 atctactaaa acaaggcaaa atgccgtgtt tatctcgtca acttgttggc gagatttttt
     2701 tgttttttat gtctgcggcc gcggtaccca attcgcccta tagtgagtcg tattagttaa
     2761 ttaacgcttc tttgaaaaga taatgtatga ttatgctttc actcatattt atacagaaac
     2821 ttgatgtttt ctttcgagta tatacaaggt gattacatgt acgtttgaag tacaactcta
     2881 gattttgtag tgccctcttg ggctagcggt aaaggtgcgc attttttcac accctacaat
     2941 gttctgttca aaagattttg gtcaaacgct gtagaagtga aagttggtgc gcatgtttcg
     3001 gcgttcgaaa cttctccgca gtgaaagata aatgatctag aaaaacagta gtggaagggt
     3061 tttagtactc tggaaacaga atctactaaa acaaggcaaa atgccgtgtt tatctcgtca
     3121 acttgttggc gagatttttt tgttttttat gtctgcggcc gcggtaccca attcgcccta
     3181 taggcactgg tctgtttagc ttgccttgtc cccgccgggt cacccggcca gcgacatgga
     3241 ggcccagaat accctccttg acagtcttga cgtgcgcagc tcaggggcat gatgtgactg
     3301 tcgcccgtac atttagccca tacatcccca tgtataatca tttgcatcca tacattttga
     3361 tggccgcacg gcgcgaagca aaaattacgg ctcctcgctg cggacctgcg agcagggaaa
     3421 cgctcccctc acagacgcgt tgaattgtcc ccacgccgcg cccctgtaga gaaatataaa
     3481 aggttaggat ttgccactga ggttcttctt tcatatactt ccttttaaaa tcttgctagg
     3541 atacagttct cacatcacat ccgaacataa acaaccatgg gtaaaaagcc tgaactcacc
     3601 gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag
     3661 ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc
     3721 ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt
     3781 gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag cgagagcctg
     3841 acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa
     3901 ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc ggccgatctt
     3961 agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg
     4021 cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac
     4081 gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac
     4141 tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac
     4201 aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac
     4261 gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc
     4321 tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc
     4381 cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct
     4441 tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca
     4501 caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat
     4561 agtggaaacc gacgccccag cactcgtccg agggcaaagg aataatcagt actgacaata
     4621 aaaagattct tgttttcaag aacttgtcat ttgtatagtt tttttatatt gtagttgttc
     4681 tattttaatc aaatgttagc gtgatttata ttttttttcg cctcgacatc atctgcccag
     4741 atgcgaagtt aagtgcgcag aaagtaatat catgcgtcaa tcgtatgtga atgctggtcg
     4801 ctatactgct gtcgattcga tactaacgcc gccatccagt gtcgaaaacg agctcgaatt
     4861 catcgatgat atcagatcca ctagtaattt ctcttctacc acgaactcga cactgtagtc
     4921 tagtggttta ggatactccc cttccactta acaagttgct tcctaaaaag aagtaacaag
     4981 gcgagggagt gacacgggtt cgaatcccgt cagtgtcagt acacagcatt ggaaataccg
     5041 ttttagtact ctggaaacag aatctactaa aacaaggcaa aatgccgtgt ttatctcgtc
     5101 aacttgttgg cgagattttt ttgtttttta tgtctgcggc cgcggtaccc aattcgccct
     5161 atagggccta tgcggccgcg gatctgccgg tctccctata gtgagtcgta ttaatttcga
     5221 taagccaggt taacctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5281 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     5341 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     5401 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5461 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     5521 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     5581 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     5641 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     5701 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     5761 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     5821 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     5881 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     5941 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     6001 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     6061 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     6121 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     6181 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6241 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6301 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6361 tgataccg
//
