LOCUS       sequence-212281-        3849 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expression of nat gene conferring resistance to nourseothricin for 
            gene deletion in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3849)
  AUTHORS   Juergens H, Varela JA, Gorter de Vries AR, Perli T, Gast VJM, 
            Gyurchev NY, Rajkumar AS, Mans R, Pronk JT, Morrissey JP, Daran JG
  TITLE     Genome editing in Kluyveromyces and Ogataea yeasts using a 
            broad-host-range Cas9/gRNA co-expression plasmid.
  JOURNAL   FEMS Yeast Res. 2018 May 1;18(3). pii: 4847887. doi: 
            10.1093/femsyr/foy012.
  PUBMED    29438517
REFERENCE   2  (bases 1 to 3849)
  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: "FEMS Yeast 
            Res."; date: "2018-05-1"; volume: "18(3). pii: 4847887. doi"; pages:
            " 10.1093/femsyr/foy012"
COMMENT     Sequence Label: pUG-natNT2
FEATURES             Location/Qualifiers
     source          1..3849
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    53..86
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        141..484
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             485..1057
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      1065..1252
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1120..1144)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    1400..1433
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(1486..1505)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1487..1505)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1592..1609)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1763..2351)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1843..1862)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2522..3313,3314..3382))
                     /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     3145..3164
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3383..3487)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3555..3573
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3611..3633)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3733..3752
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(3833..3849,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(3833..3849,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg ctgcaggtcg acaaccctta atataacttc
       61 gtataatgta tgctatacga agttattagg tctagagatc tgtttagctt gcctcgtccc
      121 cgccgggtca cccggccagc gacatggagg cccagaatac cctccttgac agtcttgacg
      181 tgcgcagctc aggggcatga tgtgactgtc gcccgtacat ttagcccata catccccatg
      241 tataatcatt tgcatccata cattttgatg gccgcacggc gcgaagcaaa aattacggct
      301 cctcgctgca gacctgcgag cagggaaacg ctcccctcac agacgcgttg aattgtcccc
      361 acgccgcgcc cctgtagaga aatataaaag gttaggattt gccactgagg ttcttctttc
      421 atatacttcc ttttaaaatc ttgctaggat acagttctca catcacatcc gaacataaac
      481 aaccatgggt accactcttg acgacacggc ttaccggtac cgcaccagtg tcccggggga
      541 cgccgaggcc atcgaggcac tggatgggtc cttcaccacc gacaccgtct tccgcgtcac
      601 cgccaccggg gacggcttca ccctgcggga ggtgccggtg gacccgcccc tgaccaaggt
      661 gttccccgac gacgaatcgg acgacgaatc ggacgacggg gaggacggcg acccggactc
      721 ccggacgttc gtcgcgtacg gggacgacgg cgacctggcg ggcttcgtgg tcgtctcgta
      781 ctccggctgg aaccgccggc tgaccgtcga ggacatcgag gtcgccccgg agcaccgggg
      841 gcacggggtc gggcgcgcgt tgatggggct cgcgacggag ttcgcccgcg agcggggcgc
      901 cgggcacctc tggctggagg tcaccaacgt caacgcaccg gcgatccacg cgtaccggcg
      961 gatggggttc accctctgcg gcctggacac cgccctgtac gacggcaccg cctcggacgg
     1021 cgagcaggcg ctctacatga gcatgccctg cccctaatct cgaggcgaat ttcttatgat
     1081 ttatgatttt tattattaaa taagttataa aaaaaataag tgtatacaaa ttttaaagtg
     1141 actcttaggt tttaaaacga aaattcttat tcttgagtaa ctctttcctg taggtcaggt
     1201 tgctttctca ggtatagcat gaggtcgctc ttattgacca cacctctacc ggcatgccga
     1261 gcaaatgcct gcaaatcgct ccccatttca cccaattgta gatatgctaa ctccagcaat
     1321 gagttgatga atctcggtgt gtattttatg tcctcagagg acaacacctg ttgtaatcga
     1381 gctctcgaga acccttaata taacttcgta taatgtatgc tatacgaagt tattaggtga
     1441 tatcagatcc actagtggcc tatgcggccg cggatctgcc ggtctcccta tagtgagtcg
     1501 tattaatttc gataagccag gttaacctgc attaatgaat cggccaacgc gcggggagag
     1561 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     1621 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     1681 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     1741 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     1801 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     1861 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     1921 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     1981 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     2041 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     2101 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     2161 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     2221 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     2281 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     2341 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     2401 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     2461 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     2521 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     2581 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     2641 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     2701 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     2761 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     2821 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     2881 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     2941 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     3001 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     3061 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     3121 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     3181 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     3241 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     3301 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     3361 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     3421 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     3481 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
     3541 cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg
     3601 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     3661 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct
     3721 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg gacatattgt
     3781 cgttagaacg cggctacaat taatacataa ccttatgtat catacacata cgatttaggt
     3841 gacactata
//
