LOCUS       sequence-192354-        5712 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 5712)
  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 5712)
  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..5712
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     gene            complement(686..1805)
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     terminator      complement(686..883)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(885..1454)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGFT
                     LREVPVDPPLTKVFPDDESDDESDAGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNRR
                     LTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFTL
                     CGLDTALYDGTASDGEQALYMSMPCP"
     promoter        complement(1462..1805)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     rep_origin      1863..2741
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     primer_bind     3356..3372
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3356..3372
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3406..3422)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3407..3423)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(3452..3470)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3491..3507)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3491..3507)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3504..3526)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3515..3531
                     /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)."
     promoter        complement(join(3539..3545,3546..3563,3564..3569))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(3722..3739)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3893..4481)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4652..5443,5444..5512))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFFHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5275..5294
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5513..5617)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5685..5703
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatcga ctacgtcgta aggccgtttc tgacagagta aaattcttga gggaactttc
      241 accattatgg gaaatggttc aagaaggtat tgacttaaac tccatcaaat ggtcaggtca
      301 ttgagtgttt tttatttgtt gtattttttt ttttttagag aaaatcctcc aatatcaaat
      361 taggaatcgt agtttcatga ttttctgtta cacctaactt tttgtgtggt gccctcctcc
      421 ttgtcaatat taatgttaaa gtgcaattct ttttccttat cacgttgagc cattagtatc
      481 aatttgctta cctgtattcc tttactatcc tcctttttct ccttcttgat aaatgtatgt
      541 agattgcgta tatagtttcg tctaccctat gaacatattc cattttgtaa tttcgtgtcg
      601 tttctattat gaatttcatt tataaagttt atgtacacct aggatccgtc gacactggat
      661 ggcggcgtta gtatcgaatc gacagcagta tagcgaccag cattcacata cgattgacgc
      721 atgatattac tttctgcgca cttaacttcg catctgggca gatgatgtcg aggcgaaaaa
      781 aaatataaat cacgctaaca tttgattaaa atagaacaac tacaatataa aaaaactata
      841 caaatgacaa gttcttgaaa acaagaatct ttttattgtc agtactaggg gcagggcatg
      901 ctcatgtaga gcgcctgctc gccgtccgag gcggtgccgt cgtacagggc ggtgtccagg
      961 ccgcagaggg tgaaccccat ccgccggtac gcgtggatcg ccggtgcgtt gacgttggtg
     1021 acctccagcc agaggtgccc ggcgccccgc tcgcgggcga actccgtcgc gagccccatc
     1081 aacgcgcgcc cgaccccgtg cccccggtgc tccggggcga cctcgatgtc ctcgacggtc
     1141 agccggcggt tccagccgga gtacgagacg accacgaagc ccgccaggtc gccgtcgtcc
     1201 ccgtacgcga cgaacgtccg ggagtccggg tcgccgtcct ccccggcgtc cgattcgtcg
     1261 tccgattcgt cgtcggggaa caccttggtc aggggcgggt ccaccggcac ctcccgcagg
     1321 gtgaagccgt ccccggtggc ggtgacgcgg aagacggtgt cggtggtgaa ggacccatcc
     1381 agtgcctcga tggcctcggc gtcccccggg acactggtgc ggtaccggta agccgtgtcg
     1441 tcaagagtgg tcattttaca tggttgttta tgttcggatg tgatgtgaga actgtatcct
     1501 agcaagattt taaaaggaag tatatgaaag aagaacctca gtggcaaatc ctaacctttt
     1561 atatttctct acaggggcgc ggcgtgggga caattcaacg cgtctgtgag gggagcgttt
     1621 ccctgctcgc aggtctgcag cgaggagccg taatttttgc ttcgcgccgt gcggccatca
     1681 aaatgtatgg atgcaaatga ttatacatgg ggatgtatgg gctaaatgta cgggcgacag
     1741 tcacatcatg cccctgagct gcgcacgtca agactgtcaa ggagggtatt ctgggcctcc
     1801 atgtcgctgg ccgggtgacc cggcggggac gaggccttaa gttcgaacgt acgagctccg
     1861 gcattgcgaa taccgctttc cacaaacatt gctcaaaagt atctctttgc tatatatctc
     1921 tgtgctatat ccctatataa cctacccatc cacctttcgc tccttgaact tgcatctaaa
     1981 ctcgacctct acatttttta tgtttatctc tagtattact ctttagacaa aaaaattgta
     2041 gtaagaacta ttcatagagt gaatcgaaaa caatacgaaa atgtaaacat ttcctatacg
     2101 tagtatatag agacaaaata gaagaaaccg ttcataattt tctgaccaat gaagaatcat
     2161 caacgctatc actttctgtt cacaaagtat gcgcaatcca catcggtata gaatataatc
     2221 ggggatgcct ttatcttgaa aaaatgcacc cgcagcttcg ctagtaatca gtaaacgcgg
     2281 gaagtggagt caggcttttt ttatggaaga gaaaatagac accaaagtag ccttcttcta
     2341 accttaacgg acctacagtg caaaaagtta tcaagagact gcattataga gcgcacaaag
     2401 gagaaaaaaa gtaatctaag atgctttgtt agaaaaatag cgctctcggg atgcattttt
     2461 gtagaacaaa aaagaagtat agattctttg ttggtaaaat agcgctctcg cgttgcattt
     2521 ctgttctgta aaaatgcagc tcagattctt tgtttgaaaa attagcgctc tcgcgttgca
     2581 tttttgtttt acaaaaatga agcacagatt cttcgttggt aaaatagcgc tttcgcgttg
     2641 catttctgtt ctgtaaaaat gcagctcaga ttctttgttt gaaaaattag cgctctcgcg
     2701 ttgcattttt gttctacaaa atgaagcaca gatgcttcgt taacaaagat atgctattga
     2761 agtgcaagat ggaaacgcag aaaatgaacc ggggatgcga cgtgcaagat tacctatgca
     2821 atagatgcaa tagtttctcc aggaaccgaa atacatacat tgtcttccgt aaagcgctag
     2881 actatatatt attatacagg ttcaaatata ctatctgttt cagggaaaac tcccaggttc
     2941 ggatgttcaa aattcaatga tgggtaacaa gtacgatcgt aaatctgtaa aacagtttgt
     3001 cggatattag gctgtatctc ctcaaagcgt attcgaatat cattgagaag ctgcagcgtc
     3061 acatcggata ataatgatgg cagccattgt agaagtgcct tttgcatttc tagtctcttt
     3121 ctcggtctag ctagttttac tacatcgcga agatagaatc ttagatcaca ctgcctttgc
     3181 tgagctggat caatagagta acaaaagagt ggtaaggcct cgttaaagga caaggacctg
     3241 agcggaagtg tatcgtacag tagacggagt atactaggta tagtctatag tccgtggaat
     3301 taattctcat gtttgacagc ttatcatcga taatccggag ctagcatgcg gccgctctag
     3361 aactagtgga tcccccgggc tgcaggaatt cgatatcaag cttatcgata ccgtcgacct
     3421 cgaggggggg cccggtaccc agcttttgtt ccctttagtg agggttaatt ccgagcttgg
     3481 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     3541 acataggagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg aggtaactca
     3601 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     3661 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     3721 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     3781 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     3841 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     3901 ggctcggccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     3961 cgacaggact ataaagatac caggcgttcc cccctggaag ctccctcgtg cgctctcctg
     4021 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     4081 tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     4141 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     4201 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     4261 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     4321 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     4381 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     4441 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     4501 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     4561 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     4621 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     4681 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actgcccgtc gtgtagataa
     4741 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     4801 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     4861 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     4921 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     4981 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     5041 ttacatgatc ccccatgttg tgaaaaaaag cggttagctc cttcggtcct ccgatcgttg
     5101 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     5161 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     5221 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     5281 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     5341 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     5401 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     5461 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     5521 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     5581 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     5641 ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
     5701 ggccctttcg tc
//
