LOCUS       sequence-199900-        5711 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multi-copy (2 micron) yeast plasmid containing a functional NatMX 
            marker cassette conferring resistance against nourseothricin. 
            Recipient plasmid for sgRNA or crRNA expression cassettes..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5711)
  AUTHORS   Verwaal R, Buiting-Wiessenhaan N, Dalhuijsen S, Roubos JA
  TITLE     CRISPR/Cpf1 enables fast and simple genome editing of Saccharomyces 
            cerevisiae.
  JOURNAL   Yeast. 2017 Sep 8. doi: 10.1002/yea.3278.
  PUBMED    28886218
REFERENCE   2  (bases 1 to 5711)
  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: "Yeast. 2017
            Sep 8. doi: 10.1002/yea.3278."
COMMENT     Sequence Label: pRN1120
FEATURES             Location/Qualifiers
     source          1..5711
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(525..544)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     644..666
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(704..722)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        790..894
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(895..963,964..1755)
                     /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"
     primer_bind     complement(1113..1132)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1926..2514
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2415..2434
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2668..2685
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2802..2823
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2838..2843,2844..2861,2862..2868)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2876..2892
                     /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)."
     primer_bind     2881..2903
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2900..2916
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2900..2916
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2937..2955
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     2983..2999
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2984..3000
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3034..3050)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3034..3050)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     rep_origin      3665..4542
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     gene            join(4600..5711,1..8)
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     promoter        4600..4943
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             4951..5520
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGFT
                     LREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNRR
                     LTVEDIEVAPEHRGHGVGRTLMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFTL
                     CGLDTALYDGTASDGEQALYMSMPCP"
     terminator      join(5522..5711,1..8)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
ORIGIN
        1 ctatactgct gtcgattcga tactaacgcc gccatccagt gtcgacggat cctaggtgta
       61 cataaacttt ataaatgaaa ttcataatag aaacgacacg aaattacaaa atggaatatg
      121 ttcatagggt agacgaaact atatacgcaa tctacataca tttatcaaga aggagaaaaa
      181 ggaggatgta aaggaataca ggtaagcaaa ttgatactaa tggctcaacg tgataaggaa
      241 aaagaattgc actttaacat taatattgac aaggaggagg gcaccacaca aaaagttagg
      301 tgtaacagaa aatcatgaaa ctatgattcc taatttatat attggaggat tttctctaaa
      361 aaaaaaaaaa tacaacaaat aaaaaacact caatgacctg accatttgat ggagtttaag
      421 tcaatacctt cttgaaccat ttcccataat ggtgaaagtt ccctcaagaa ttttactctg
      481 tcagaaacgg ccttaacgac gtagtcgata tggtgcactc tcagtacaat ctgctctgat
      541 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
      601 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
      661 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
      721 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
      781 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
      841 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
      901 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
      961 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     1021 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     1081 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     1141 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     1201 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     1261 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     1321 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     1381 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     1441 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     1501 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     1561 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     1621 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     1681 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     1741 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     1801 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     1861 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     1921 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     1981 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     2041 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     2101 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     2161 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     2221 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     2281 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     2341 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     2401 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     2461 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     2521 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     2581 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     2641 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     2701 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     2761 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
     2821 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
     2881 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gctcgaaatt
     2941 aaccctcact aaagggaaca aaagctggta ccgggccccc cctcgaggtc gacggtatcg
     3001 ataagcttga tatcgaattc ctgcagcccg ggggatccac tagttctaga gcggccgcat
     3061 gctagctccg gattatcgat gataagctgt caaacatgag aattaattcc acggactata
     3121 gactatacct agtatactcc gtctactgta cgatacactt ccgctcaggt ccttgtcctt
     3181 taacgaggcc ttaccactct tttgttactc tattgatcca gctcagcaaa ggcagtgtga
     3241 tctaagattc tatcttcgcg atgtagtaaa actagctaga ccgagaaaga gactagaaat
     3301 gcaaaaggca cttctacaat ggctgccatc attattatcc gatgtgacgc tgcagcttct
     3361 caatgatatt cgaatacgct ttgaggagat acagcctaat atccgacaaa ctgttttaca
     3421 gatttacgat cgtacttgtt acccatcatt gaattttgaa catccgaacc tgggagtttt
     3481 ccctgaaaca gatagtatat ttgaacctgt ataataatat atagtctagc gctttacgga
     3541 agacaatgta tgtatttcgg ttcctggaga aactattgca tctattgcat aggtaatctt
     3601 gcacgtcgca tccccggttc attttctgcg tttccatctt gcacttcaat agcatatctt
     3661 tgttaacgaa gcatctgtgc ttcattttgt agaacaaaaa tgcaacgcga gagcgctaat
     3721 ttttcaaaca aagaatctga gctgcatttt tacagaacag aaatgcaacg cgaaagcgct
     3781 attttaccaa cgaagaatct gtgcttcatt tttgtaaaac aaaaatgcaa cgcgagagcg
     3841 ctaatttttc aaacaaagaa tctgagctgc atttttacag aacagaaatg caacgcgaga
     3901 gcgctatttt accaacaaag aatctatact tcttttttgt tctacaaaaa tgcatcccga
     3961 gagcgctatt tttctaacaa agcatcttag attacttttt ttctcctttg tgcgctctat
     4021 aatgcagtct cttgataact ttttgcactg taggtccgtt aaggttagaa gaaggctact
     4081 ttggtgtcta ttttctcttc cataaaaaaa gcctgactcc acttcccgcg tttactgatt
     4141 actagcgaag ctgcgggtgc attttttcaa gataaaggca tccccgatta tattctatac
     4201 cgatgtggat tgcgcatact ttgtgaacag aaagtgatag cgttgatgat tcttcattgg
     4261 tcagaaaatt atgaacggtt tcttctattt tgtctctata tactacgtat aggaaatgtt
     4321 tacattttcg tattgttttc gattcactct atgaatagtt cttactacaa tttttttgtc
     4381 taaagagtaa tactagagat aaacataaaa aatgtagagg tcgagtttag atgcaagttc
     4441 aaggagcgaa aggtggatgg gtaggttata tagggatata gcacagagat atatagcaaa
     4501 gagatacttt tgagcaatgt ttgtggaagc ggtattcgca atgccggagc tcgtacgttc
     4561 gaacttaagg cctcgtcccc gccgggtcac ccggccagcg acatggaggc ccagaatacc
     4621 ctccttgaca gtcttgacgt gcgcagctca ggggcatgat gtgactgtcg cccgtacatt
     4681 tagcccatac atccccatgt ataatcattt gcatccatac attttgatgg ccgcacggcg
     4741 cgaagcaaaa attacggctc ctcgctgcag acctgcgagc agggaaacgc tcccctcaca
     4801 gacgcgttga attgtcccca cgccgcgccc ctgtagagaa atataaaagg ttaggatttg
     4861 ccactgaggt tcttctttca tatacttcct tttaaaatct tgctaggata cagttctcac
     4921 atcacatccg aacataaaca accatgtaaa atgaccactc ttgacgacac ggcttaccgg
     4981 taccgcacca gtgtcccggg ggacgccgag gccatcgagg cactggatgg gtccttcacc
     5041 accgacaccg tcttccgcgt caccgccacc ggggacggct tcaccctgcg ggaggtgccg
     5101 gtggacccgc ccctgaccaa ggtgttcccc gacgacgaat cggacgacga atcggacgac
     5161 ggggaggacg gcgacccgga ctcccggacg ttcgtcgcgt acggggacga cggcgacctg
     5221 gcgggcttcg tggtcgtctc gtactccggc tggaaccgcc ggctgaccgt cgaggacatc
     5281 gaggtcgccc cggagcaccg ggggcacggg gtcgggcgca cgttgatggg gctcgcgacg
     5341 gagttcgccc gcgagcgggg cgccgggcac ctctggctgg aggtcaccaa cgtcaacgca
     5401 ccggcgatcc acgcgtaccg gcggatgggg ttcaccctct gcggcctgga caccgccctg
     5461 tacgacggca ccgcctcgga cggcgagcag gcgctctaca tgagcatgcc ctgcccctag
     5521 tactgacaat aaaaagattc ttgttttcaa gaacttgtca tttgtatagt ttttttatat
     5581 tgtagttgtt ctattttaat caaatgttag cgtgatttat attttttttc gcctcgacat
     5641 catctgccca gatgcgaagt taagtgcgca gaaagtaata tcatgcgtca atcgtatgtg
     5701 aatgctggtc g
//
