LOCUS       sequence-386073-        5539 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  nourseothricin (NAT) resistance genes codon-optimized for R. 
            toruloides and expressed under pXYL and tNOS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5539)
  AUTHORS   Bonturi N, Pinheiro MJ, de Oliveira PM, Rusadze E, Eichinger T, 
            Liudziute G, De Biaggi JS, Brauer A, Remm M, Miranda EA, 
            Ledesma-Amaro R, Lahtvee PJ
  TITLE     Development of a dedicated Golden Gate Assembly Platform (RtGGA) for
            Rhodotorula toruloides.
  JOURNAL   Metab Eng Commun. 2022 May 23;15:e00200. doi: 
            10.1016/j.mec.2022.e00200. eCollection 2022 Dec.
  PUBMED    35662893
REFERENCE   2  (bases 1 to 5539)
  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: "Metab Eng 
            Commun."; date: "2022-05-23"; volume: "15:e00200. doi"; pages: " 
            10.1016/j.mec.2022.e00200. eCollection 2022 Dec"
COMMENT     Sequence Label: (B) NAT marker (R) (SBE173)
FEATURES             Location/Qualifiers
     source          1..5539
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     126..148
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     140..157
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     141..157
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     164..183
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        164..182
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1020..1592
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRATATGDGF
                     TLREVPMDPPLTKVFPDDESDDESDDGEDGDPGSRTFVAYGDDGDLAGFVVVLYSGWNR
                     RLTVEDIEVAPEHRGYGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHTYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     primer_bind     complement(1934..1950)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1934..1950)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1947..1969)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1958..1974
                     /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(1982..1988,1989..2006,2007..2012))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2027..2048
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(2336..2924)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2416..2435)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3095..3886,3887..3955))
                     /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     3718..3737
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             complement(3973..4767)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4084..4103)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4694..4713
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     rep_origin      complement(5111..5539)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5221..5242)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5433..5452
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
       61 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
      121 gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa ttgtaatacg actcactata
      181 gggcgaattg ggccctctag atgcatgctc gagcggccgc cagtgtgatg gatatctgca
      241 gaattcgccc ttgatgtggg tctccaggtt gtccgtattc tacatcgacg aagcagggcg
      301 gggcggacag gcgtttgtcg ttgcgcgagg agaacaaggg cggtagagtt gttcttccgc
      361 cagctttgtc gcccacgctc gtctgcaacg tccgtcgtca ttctaccctc cactttgacg
      421 gccttctcaa gcgcgagggg cacgctccga cgccggggtg gagcagggag gggtcgctgg
      481 agctcgctcg aaatggtgga cagtgcgctg cccgcccagg agagacggaa gagacaggaa
      541 gcgccggtag aaacccgggc ggaggaggaa gaggaggttg cagaggaaac ccggtctgtc
      601 ccgctcgctg gctgaaaaag tgcactgggg ctgcttgatc gatgaggagg agacgcaggc
      661 tggacagggg tccgacgaag gaagcgagcc gacgccatgc gggccgacag acgcttgtcc
      721 acttcgctgg cgtctctctc tcgctccttc tccctcccag actcagccaa actcagtctg
      781 tgagtagctt tcgacgcatg cgagcgacag cactcaccca ggctcgctca ctgacccgct
      841 tcactcctgc ccgtcctccc cccgcttcgc accgctgcac gtcatctcct cgcgtccccg
      901 gttcttcctc tcgcccgtgc aggtgcgtgg catcgtatac actgttttgc taatttctcg
      961 aggacttcag ctaacccgcg tacacttacc cgcacgcaga atacacgcca tgtcgatcaa
     1021 tgggtaccac tcttgacgac acggcttacc ggtaccgcac cagtgtcccg ggggacgccg
     1081 aggccatcga ggcactggat gggtccttca ccaccgacac tgtcttccgc gccaccgcca
     1141 ccggggacgg cttcaccctg cgggaggtgc cgatggaccc gcccctgacc aaggtgttcc
     1201 ccgacgacga atcggacgac gaatcggacg acggggagga cggcgacccg ggctcccgga
     1261 cgttcgtcgc gtacggggac gacggcgacc tggcgggctt cgtggtcgtc ttgtactccg
     1321 gctggaaccg ccggctgacc gtcgaggaca tcgaggtcgc cccggagcac cgggggtacg
     1381 gggtcgggcg cgcgttgatg gggctcgcga cggagttcgc ccgcgagcgg ggcgccgggc
     1441 acctctggct ggaggtcacc aacgttaacg caccggctat ccacacgtac cggcggatgg
     1501 ggttcaccct ctgcggcctg gacaccgccc tgtacgacgg caccgcctcg gacggcgagc
     1561 aggcgctcta catgagcatg ccctgcccct aacgttcaaa catttggcaa taaagtttct
     1621 taagattgaa tcctgttgcc ggtcttgcga tgatcatcat ataatttctg ttgaattacg
     1681 ttaagcatgt aataattaac atgtaatgca tgacgttatt tatgagatgg gtttttatga
     1741 ttagagtccc gcaattatac atttaatacg cgatagaaaa caaaatatag cgcgcaaact
     1801 aggataaatt atcgcgcgcg gtgtcatcta tgttactaga tcgggacggt gagaccgcat
     1861 aagggcgaat tccagcacac tggcggccgt tactagtgga tccgagctcg gtaccaagct
     1921 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     1981 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     2041 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     2101 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     2161 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     2221 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     2281 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     2341 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     2401 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     2461 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     2521 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     2581 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     2641 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     2701 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     2761 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     2821 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     2881 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     2941 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     3001 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     3061 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     3121 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     3181 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     3241 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     3301 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     3361 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     3421 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     3481 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     3541 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     3601 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     3661 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     3721 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     3781 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     3841 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     3901 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     3961 tcctttttca attcagaaga actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc
     4021 gggagcggcg ataccgtaaa gcacgaggaa gcggtcagcc cattcgccgc caagctcttc
     4081 agcaatatca cgggtagcca acgctatgtc ctgatagcgg tccgccacac ccagccggcc
     4141 acagtcgatg aatccagaaa agcggccatt ttccaccatg atattcggca agcaggcatc
     4201 gccatgggtc acgacgagat cctcgccgtc gggcatgcgc gccttgagcc tggcgaacag
     4261 ttcggctggc gcgagcccct gatgctcttc gtccagatca tcctgatcga caagaccggc
     4321 ttccatccga gtacgtgctc gctcgatgcg atgtttcgct tggtggtcga atgggcaggt
     4381 agccggatca agcgtatgca gccgccgcat tgcatcagcc atgatggata ctttctcggc
     4441 aggagcaagg tgggatgaca ggagatcctg ccccggcact tcgcccaata gcagccagtc
     4501 ccttcccgct tcagtgacaa cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag
     4561 ccacgatagc cgcgctgcct cgtcctgcag ttcattcagg gcaccggaca ggtcggtctt
     4621 gacaaaaaga accgggcgcc cctgcgctga cagccggaac acggcggcat cagagcagcc
     4681 gattgtctgt tgtgcccagt catagccgaa tagcctctcc acccaagcgg ccggagaacc
     4741 tgcgtgcaat ccatcttgtt caatcatgcg aaacgatcct catcctgtct cttgatcaga
     4801 tcttgatccc ctgcgccatc agatccttgg cggcaagaaa gccatccagt ttactttgca
     4861 gggcttccca accttaccag agggcgcccc agctggcaat tccggttcgc ttgctgtcca
     4921 taaaaccgcc cagtctagct atcgccatgt aagcccactg caagctacct gctttctctt
     4981 tgcgcttgcg ttttcccttg tccagatagc ccagtagctg acattcatcc ggggtcagca
     5041 ccgtttctgc ggactggctt tctacgtgtt ccgcttcctt tagcagccct tgcgccctga
     5101 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     5161 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     5221 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     5281 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     5341 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     5401 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     5461 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     5521 gcgccgctac agggcgcgt
//
