LOCUS       sequence-386125-        4513 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  integration cassette for ku70 deletion and expression of NAT gene 
            under pLDP1 for characterization.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4513)
  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 4513)
  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: ku70 insUP+pLDP1+NAT+tNOS+ku70 insD (SBE193)
FEATURES             Location/Qualifiers
     source          1..4513
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1055..1627
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPMDPPLTKVFPDDESDDESDDGEDGDPGSRTFVAYGDDGDLAGFVVVLYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHTYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     primer_bind     complement(2452..2471)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2453..2471)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(2582..3241)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3156..3175
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(3242..3344)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      3445..4033
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3934..3953
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4187..4204
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4463..4481
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4463..4480
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gacttggtac ggagccgcct cctccacctc agcaaccaac ctaccagtcg caccccggat
       61 cgcagtctag tcttccgccg cttcccgctc tcccttcttc ctcgatgccg cctcctccgc
      121 ttcctcggcc caaggcgcct cagagcgccg catcctccgc ctcaacgctt ccgcctccgc
      181 cattctaggc gatgccggga ggaaccgtgc tgaacggaca aggtacggcc ccggcggcag
      241 tccaggcgaa cggggtgcag cagtgaggag cgaacagtat gcatagctgg atcgtcgcat
      301 gcgtcttagt gtcttgcttt cttgttggaa aatcgttgga atcgttggac cttgttgtag
      361 cctctctcac gcaacgactg gcttgtttga cgtctcttca ttgcgtgggg agagtgaagc
      421 gatgtttgca aagtaggaca caacaagcga acggactact gcgaacgtac acaggcgaga
      481 gcgatcgggg gcgaaagtca gtcgtcgtcc tcctcatcgc cgacgatgaa ggtcacgcct
      541 ctgtgactcg gtacggagag agagagcgtt gggttgggtt ggggattgtg gcgagcgaag
      601 ggcgacccag cagccaggga gagggagttg gtctggatgc aaaccatgcg catctcctct
      661 cgcagttgaa tcgtttttcc cgctctgccc tcgctctctc ttccttctgc tctttactcg
      721 ctcacgaaca acaacgagcc acacagcgtg agcacacacc gctgcactca ctcgctgtca
      781 cggaccgcag ctcaccctta tcgtcactcc ctctcccacc gcacagcctc actccctctc
      841 tcgctctccc tcacaagcac aacacacggc acactcgcac gcacactcgc acgcaatggc
      901 caccgtcaac gagaagcagc ccgccaccga cgcgcccctc gcgcacgata ccgccatcca
      961 ccgcgtgcgt ccccatccct cccactgtca tcctcgtgaa acccgctcac ccgttcgcaa
     1021 gcacacacgc aggtgtcgga ctaccccatg atcaatgggt accactcttg acgacacggc
     1081 ttaccggtac cgcaccagtg tcccggggga cgccgaggcc atcgaggcac tggatgggtc
     1141 cttcaccacc gacaccgtct tccgcgtcac cgccaccggg gacggcttca ccctgcggga
     1201 ggtgccgatg gacccgcccc tgaccaaggt gttccccgac gacgaatcgg acgacgaatc
     1261 ggacgacggg gaggacggcg acccgggctc ccggacgttc gtcgcgtacg gggacgacgg
     1321 cgacctggcg ggcttcgtgg tcgtcttgta ctccggctgg aaccgccggc tgaccgtcga
     1381 ggacatcgag gtcgccccgg agcaccgggg gcacggggtc gggcgcgcgt tgatggggct
     1441 cgcgacggag ttcgcccgcg agcggggcgc cgggcacctc tggctggagg tcaccaacgt
     1501 taacgcaccg gctatccaca cgtaccggcg gatggggttc accctctgcg gcctggacac
     1561 cgccctgtac gacggcaccg cctcggacgg cgagcaggcg ctctacatga gcatgccctg
     1621 cccctaacgt tcaaacattt ggcaataaag tttcttaaga ttgaatcctg ttgccggtct
     1681 tgcgatgatt atcatataat ttctgttgaa ttacgttaag catgtaataa ttaacatgta
     1741 atgcatgacg ttatttatga gatgggtttt tatgattaga gtcccgcaat tatacattta
     1801 atacgcgata gaaaacaaaa tatagcgcgc aaactaggat aaattatcgc gcgcggtgtc
     1861 atctatgtta ctagatcggg acggatgtgg cgaccgggcg cgcgtttctt gccctcggcg
     1921 caagtgggca ttcgctcagg cgggcagggt tcttcggcgg tagttcacgc gcatcagtac
     1981 gtgccgcgcc ctttcttcgc cttacttatt gtgagctgac gcactcttgc acagtaacgt
     2041 acaaagcgac tcggaagacg acgacggctt tgcgcacgtc cacgccaagc acgcctacga
     2101 gaattttgac gaggttgcgc cgcacgagat tgacccgtac gtgccgctgt ctctttcgaa
     2161 tgcatcacaa gctcgagctg atgccggccg caacacccag cctccgcgat gttgcggctt
     2221 cgttcctcct cgaaatgttc gaggaactct ggcgaattat gaagccgaac gagtatcgcg
     2281 gcgacaacaa aagcgacctc ctcgctcaca tccaccgaca ctgcctcccc tcgctcctca
     2341 agcacatacc gcctcggatc caccacaccg tcgcgcccga aacggtctac ccattcctgt
     2401 agtcttctta attaagacgt cagaattctc gaggcggccg catgtgcgtc tccctatagt
     2461 gagtcgtatt aatttcgcgg gcggaacccc tatttgttta tttttctaaa tacattcaaa
     2521 tatgtatccg ctcatgagta gcaccaggcg tttaagggca ccaataactg ccttaaaaaa
     2581 attacgcccc gccctgccac tcatcgcagt actgttgtaa ttcattaagc attctgccga
     2641 catggaagcc atcacaaacg gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt
     2701 cgccttgcgt ataatatttg cccatggtga aaacgggggc gaagaagttg tccatattgg
     2761 ccacgtttaa atcaaaactg gtgaaactca cccagggatt ggctgagacg aaaaacatat
     2821 tctcaataaa ccctttaggg aaataggcca ggttttcacc gtaacacgcc acgtcttgcg
     2881 aatatatgtg tagaaactgc cggaaatcgt cgtggtattc actccagagc gatgaaaacg
     2941 tttcagtttg ctcatggaaa acggtgtaac aagggtgaac actatcccat atcaccagct
     3001 caccgtcttt cattgccata cggaattccg gatgagcatt catcaggcgg gcaagaatgt
     3061 gaataaaggc cggataaaac ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa
     3121 tatccagctg aacggtctgg ttataggtac attgagcaac tgactgaaat gcctcaaaat
     3181 gttctttacg atgccattgg gatatatcaa cggtggtata tccagtgatt tttttctcca
     3241 ttttagcttc cttagctcct ggaaatctcg ataactcaaa aaatacgccc ggtagtgatc
     3301 ttatttcatt atggtgaaag ttggaacctc ttacgtgccg atcaacgtct cattttcgcc
     3361 aaaagttgtc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     3421 cgtagaaaag atcgaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     3481 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     3541 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     3601 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     3661 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     3721 ctcaagacga tagttgccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     3781 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     3841 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     3901 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     3961 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     4021 gagcctatgg aaaaacgcca gcaatgcggc ctttttacgg ttcctggcct tttgctggcc
     4081 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     4141 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     4201 cgaggaagcc gaaaaatcaa taatcagaca acaagatgtg cgaactcgat attttacacg
     4261 actctcttta ccaattctgc cccgaattac acttaaaacg actcaacagc ttaacgttgg
     4321 cttgccacgc attacttgac tgtaaaactc tcactcttac cgaacttggc cgtaacctgc
     4381 caaccaaagc gagaacaaaa cataacatca aacgaatcga ccgattgtta ggtaatcgtc
     4441 acctgcagga aggtttaaac gcatttaggt gacactatag aagtgtgtat cgctcgaggg
     4501 atccgaattc gaa
//
