LOCUS       sequence-386086-        5796 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  geneticin (G418) 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 5796)
  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 5796)
  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) G418 marker (R) (SBE 137)
FEATURES             Location/Qualifiers
     source          1..5796
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        242..260
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2168..2187)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2169..2187)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2194..2210)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2203..2225)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             2348..2650
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     2529..2548
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2999..3793
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2999..3793
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3053..3072)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3663..3682
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             complement(join(4043..4834,4835..4903))
                     /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     4666..4685
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5027..5615
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5516..5535
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctca
      241 gaattaaccc tcactaaagg gactagtcct gcaggtttaa acgaattcgc ccttagagca
      301 ggtctccaca cccgatctag taacatagat gacaccgcgc gcgataattt atcctagttt
      361 gcgcgctata ttttgttttc tatcgcgtat taaatgtata attgcgggac tctaatcata
      421 aaaacccatc tcataaataa cgtcatgcat tacatgttaa ttattacatg cttaacgtaa
      481 ttcaacagaa attatatgat aatcatcgca agaccggcaa caggattcaa tcttaagaaa
      541 ctttattgcc aaatgtttga acgctagaag aactcgtcga gcatgaggtg gaactggagc
      601 ttgttcatgt ccgggttgtc gatgccgtac ttctggaaga ggcgcttctg gagcgacggc
      661 gagaactcgc cgaggcagtt ccagaggatg gcgaggtcct ggtagcggtc ggcgatgccg
      721 acgcggccga cgtcgatgca gccgatgagc ttgccctcgt cgaagatgag gttgtcgagc
      781 gagaagtcgc cgtgggtgac gaccgagtcc ggcgagaacg ggaggagctt gtgcatctcc
      841 ttccagacct gctcgaccgg ccagccgttg cgctcgtcgt cgaagtccga ggcgtcgacg
      901 aggccgttgt tcatgcgcga ctgggcctgg gcgaggcgga agacgcggtc cgagttgaac
      961 gggcagttgc agaccgggat cgagtggagg cggcggagga agacggcgag ggcgtcgacg
     1021 atgttctcgc ccgagtccgg gtactcctcg aggacctgga aggcggtctt gcccgggatg
     1081 gcggtggtga ggagccaggc gtcgtccggg gtgcggatga agtgcttgat ggtcgggagc
     1141 ggcatgaact cggtgagcca gttgaggcgg accatctcgt cggtgacgtc gttggcgacc
     1201 gagcccttgc cgtgcttgag gaagagctcc ggggcgtccg gcttgccgta gaggcggtag
     1261 atggtggcgc ccgactggcc gacgttgtcg cgggcccact tgtagccgta gaggtcggcg
     1321 tccatgttcg agttgaggcg cgggcgcgag acgtgggtct tctccttgcc catcgacatg
     1381 gcgtgtattc tgcgtgcggg taagtgtacg cgggttagct gaagtcctcg agaaattagc
     1441 aaaacagtgt atacgatgcc acgcacctgc acgggcgaga ggaagaaccg gggacgcgag
     1501 gagatgacgt gcagcggtgc gaagcggggg gaggacgggc aggagtgaag cgggtcagtg
     1561 agcgagcctg ggtgagtgct gtcgctcgca tgcgtcgaaa gctactcaca gactgagttt
     1621 ggctgagtct gggagggaga aggagcgaga gagagacgcc agcgaagtgg acaagcgtct
     1681 gtcggcccgc atggcgtcgg ctcgcttcct tcgtcggacc cctgtccagc ctgcgtctcc
     1741 tcctcatcga tcaagcagcc ccagtgcact ttttcagcca gcgagcggga cagaccgggt
     1801 ttcctctgca acctcctctt cctcctccgc ccgggtttct accggcgctt cctgtctctt
     1861 ccgtctctcc tgggcgggca gcgcacggtc caccatttcg agcgagctcc agcgacccct
     1921 ccctgctcca ccccggcgtc ggagcgtgcc cctcgcgctt gagaaggccg tcaaagtgga
     1981 gggtagaatg acgacggacg ttgcagacga gcgtgggcga caaagctggc ggaagaacaa
     2041 ctctaccgcc cttgttctcc tcgcgcaacg acaaacgcct gtccgccccg ccctgcttcg
     2101 tcgatgtaga atacggacaa cctggagacc tgctaagggc gaattcgcgg ccgctaaatt
     2161 caattcgccc tatagtgagt cgtattacaa ttcactggcc gtcgttttac aacgtcgtga
     2221 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     2281 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctata
     2341 cgtacggcag tttaaggttt acacctataa aagagagagc cgttatcgtc tgtttgtgga
     2401 tgtacagagt gatattattg acacgccggg gcgacggatg gtgatccccc tggccagtgc
     2461 acgtctgctg tcagataaag tctcccgtga actttacccg gtggtgcata tcggggatga
     2521 aagctggcgc atgatgacca ccgatatggc cagtgtgccg gtctccgtta tcggggaaga
     2581 agtggctgat ctcagccacc gcgaaaatga catcaaaaac gccattaacc tgatgttctg
     2641 gggaatataa atgtcaggca tgagattatc aaaaaggatc ttcacctaga tccttttcac
     2701 gtagaaagcc agtccgcaga aacggtgctg accccggatg aatgtcagct actgggctat
     2761 ctggacaagg gaaaacgcaa gcgcaaagag aaagcaggta gcttgcagtg ggcttacatg
     2821 gcgatagcta gactgggcgg ttttatggac agcaagcgaa ccggaattgc cagctggggc
     2881 gccctctggt aaggttggga agccctgcaa agtaaactgg atggctttct tgccgccaag
     2941 gatctgatgg cgcaggggat caagctctga tcaagagaca ggatgaggat cgtttcgcat
     3001 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     3061 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     3121 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     3181 agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     3241 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     3301 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     3361 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     3421 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     3481 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg
     3541 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     3601 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     3661 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     3721 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     3781 cgagttcttc tgaattatta acgcttacaa tttcctgatg cggtattttc tccttacgca
     3841 tctgtgcggt atttcacacc gcatcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     3901 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga ttatcaaaaa
     3961 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     4021 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     4081 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     4141 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     4201 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     4261 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     4321 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     4381 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     4441 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     4501 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     4561 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     4621 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     4681 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     4741 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     4801 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     4861 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     4921 attattgaag catttatcag ggttattgtc tcatgaccaa aatcccttaa cgtgagtttt
     4981 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     5041 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     5101 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     5161 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     5221 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     5281 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     5341 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     5401 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     5461 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     5521 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     5581 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     5641 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     5701 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     5761 ccgagcgcag cgagtcagtg agcgaggaag cggaag
//
