LOCUS       sequence-386112-        4438 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  hygromycin (HYG) resistance genes codon-optimized for R. toruloides 
            and expressed under pXYL and tGPD.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4438)
  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 4438)
  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) HYG marker(R) (SBE264)
FEATURES             Location/Qualifiers
     source          1..4438
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(2273..2289)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2273..2289)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2286..2308)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(2461..3049)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2541..2560)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(3166..3190)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(3290..4078)
                     /codon_start=1
                     /gene="bla(M)"
                     /product="beta-lactamase lacking the signal sequence"
                     /label=bla(M)
                     /note="allows cytosolic expression of beta-lactamase"
                     /translation="PETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMST
                     FKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMS
                     DNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMATT
                     LRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAA
                     LGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW"
     primer_bind     3913..3932
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4152..4256)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4355..4377
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4369..4386
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4370..4386
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 tctctgggtc tcgaggttgt ccgtattcta catcgacgaa gcagggcggg gcggacaggc
       61 gtttgtcgtt gcgcgaggag aacaagggcg gtagagttgt tcttccgcca gctttgtcgc
      121 ccacgctcgt ctgcaacgtc cgtcgtcatt ctaccctcca ctttgacggc cttctcaagc
      181 gcgaggggca cgctccgacg ccggggtgga gcagggaggg gtcgctggag ctcgctcgaa
      241 atggtggacc gtgcgctgcc cgcccaggag agacggaaga gacaggaagc gccggtagaa
      301 acccgggcgg aggaggaaga ggaggttgca gaggaaaccc ggtctgtccc gctcgctggc
      361 tgaaaaagtg cactggggct gcttgatcga tgaggaggag acgcaggctg gacaggggtc
      421 cgacgaagga agcgagccga cgccatgcgg gccgacagac gcttgtccac ttcgctggcg
      481 tctctctctc gctccttctc cctcccagac tcagccaaac tcagtctgtg agtagctttc
      541 gacgcatgcg agcgacagca ctcacccagg ctcgctcact gacccgcttc actcctgccc
      601 gtcctccccc cgcttcgcac cgctgcacgt catctcctcg cgtccccggt tcttcctctc
      661 gcccgtgcag gtgcgtggca tcgtatacac tgttttgcta atttctcgag gacttcagct
      721 aacccgcgta cacttacccg cacgcagaat acacgccatg tcgccacatg aagaaacctg
      781 agcttaccgc tacctcggtc gagaagttcc ttatcgagaa gttcgactcg gtgtcggacc
      841 tcatgcaact ctcggaaggc gaggaatcgc gcgctttctc gttcgacgtt ggcggtcgcg
      901 gctacgtcct tcgcgtcaac tcgtgcgcag acggattcta caaggaccgc tacgtctacc
      961 gacattttgc atcggccgca cttccaatcc ctgaggtcct cgacattggc gagttctcgg
     1021 agtcactcac ctactgcatc tcgcgccgcg cccaaggcgt gacactccag gatcttccag
     1081 aaacggaact ccctgccgtc cttcagccgg tcgcagaagc gatggatgcc atcgctgccg
     1141 cagacctttc gcagacctcg ggctttggac cattcggtcc gcagggcatc ggccagtaca
     1201 ccacatggcg cgattttatc tgcgccatcg ccgacccgca cgtctaccaa tggcagaccg
     1261 ttatggacga aaccgtctcg gcatcggtcg cccaggccct cgacgaactt atgctctggg
     1321 ctgaggattg cccagaggtc cgccatctcg tccatgccga cttcggatcg aacaatgtcc
     1381 tcaccgacaa cggacgcatc accgccgtca ttgactggtc tgaagcaatg ttcggcgact
     1441 cgcaatacga agtcgctaac atttttttct ggcgaccttg gcttgcttgc atggagcagc
     1501 agactcgcta cttcgaacgc cgacatccgg aactcgccgg ctcgccgcgc ctccgcgcct
     1561 acatgctccg catcggcctc gaccaactct accaatcgct tgtcgacgga aactttgacg
     1621 atgccgcatg ggcacaaggc cgatgcgacg ccattgtccg ctcgggcgcc ggaaccgtcg
     1681 gtcgcaccca gattgctcgc cgatctgctg ctgtctggac cgacggatgc gtcgaggtcc
     1741 tcgctgattc gggaaaccga cgaccttcga cccgacctcg cgctaaagag tagcgagctc
     1801 cccggatcca tcaccaaggg cgtcaacttt atccagcagc ccaagctctg actcgctgac
     1861 ccggctacgg gtaccgcaaa gttgtagtta gcttggcttc ctgtctctct cttcgtcgcg
     1921 ctcgagtggt tttcggtgca aaaggacgtc cgaatcagtt cgtagtatgc tcgtcgcact
     1981 cgagaggatg ctggcttgcg ccgatgagca acgggaacga aggcggaggc agacttaggc
     2041 tagcgcggac gaggggaagc gagaggcaga cgtctatctc tcgactccgt catctctccc
     2101 gcatcgtcaa cgcacaggct gtgaactgaa ttttgtgcat tcactcaaac tgcgcagtgg
     2161 aagaagcgaa gaggctaggc gagaggggcg ggagtctaag tgtggagacc cagagaaggc
     2221 taggtggagg ctcagtgatg ataagtctgc gatggtggat gcatgtgtca tggtcatagc
     2281 tgtttcctgt gtgaaattgt tatccgctca gagggcacaa tcctattccg cgctatccga
     2341 caatctccaa gacattaggt ggagttcagt tcggcgtatg gcatatgtcg ctggaaagaa
     2401 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     2461 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     2521 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     2581 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     2641 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     2701 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     2761 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     2821 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     2881 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     2941 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     3001 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     3061 gatcttttct acggggtctg acgctctatt caacaaagcc gccgtcccgt caagtcagcg
     3121 taaatgggta gggggcttca aatcgtcctc gtgataccaa ttcggagcct gcttttttgt
     3181 acaaacttgt tgataatggc aattcaagga tcttcaccta gatcctttta aattaaaaat
     3241 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     3301 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     3361 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     3421 tgataccgcg agagccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     3481 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     3541 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     3601 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     3661 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     3721 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     3781 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     3841 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     3901 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     3961 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     4021 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     4081 gagcaaaaac aggaaggcaa aatgccgcaa aaaaagggaa taagggcgac acggaaatgt
     4141 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     4201 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     4261 tttccccgaa aagtgccaga tacctgaaac aaaacccatc gtacggccaa ggaagtctcc
     4321 aataactgtg atccaccaca agcgccaggg ttttcccagt cacgacgttg taaaacgacg
     4381 gccagtcatg cataatccgc acgcatctgg aataaggaag tgccattccg cctgacct
//
