LOCUS       sequence-386134-        5455 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  integration cassette for ku70 deletion and expression of NAT gene 
            under pPGI for characterization.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5455)
  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 5455)
  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+pPGI+NAT+tNOS+ku70 insD (SBE217)
FEATURES             Location/Qualifiers
     source          1..5455
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1997..2569
                     /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(3394..3413)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3395..3413)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(3524..4183)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4098..4117
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4184..4286)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      4387..4975
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4876..4895
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5129..5146
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5405..5423
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     5405..5422
                     /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 ggtgcatggc
      541 cgtctgccat ctcgacatcg ccgttcgcct gttgtgcgtc gccgttggtt gcgggcggtg
      601 gagcggacgt cgacggtccc gccgcgggcg cttctgcggg tggctgggtc gagatctcgg
      661 tcattgtagg cgtgcaatca gctcagcaac gtgccacaag cagcggtgcg agatggaagc
      721 gctgtcctgg tcgaaggaga tgaagcgtct cgccagaacc gctcacctcg cccacaacag
      781 cacgctcggg ctcggctggc gactgcttag acttggttgg ctgcttcaga gacgcccagc
      841 accgacagaa acggcgcgga acggtcgagt gcaccgctca cactcttccc tctctttcgc
      901 atcgaagcgg cgacacggcg gcggatcgct caagggattg tgtgtagctc gcaaaggcag
      961 tcagtggagg ataggaaccc ccctggctaa accggactct tctcgattcg ctcgatcccg
     1021 acccgtcctt cgcctccagt ggatgagctc gagtggccgg taacgagcgg tggacaggtc
     1081 atgcgctttc attgtctcgg tggtgcgttc gaattcgttc agcctgcccg ggaacgcaag
     1141 ctgaccgcgc tcttgcagtt ggatccatcg gttccctcat cgcgaccaac ctcgcccagc
     1201 ttccattctc ccaagtccgc tgcattcccg agacgaagcg tctctcgctc gaacaaagtc
     1261 cgccttccat tcaccctcgt acgcattgtc aaacgaggtg actgacgcgc tgatctatcg
     1321 cagtcgacat cctgtaccgc ctgaccggca acgcgattcg ggcgaccaag atccgccgcg
     1381 aactgtacgt ttcgtcttcc ctctcctgct ctccgaccgt gcactgacac cgtcttccgc
     1441 ccatgcacag cctcgagcgg gacatccaag acgaggatac cgccgcctat ttctacgcct
     1501 tcaaggagca acacctcggc tcttcgctgc acgagctcga gcagcaccaa ggctcgtcgt
     1561 ccgacccgca gcagccgcca catcaccaac tccagcttca gcaggacggc ctcgcaccgg
     1621 tcgagagtcc tgcggtcaag atgcgggact tgaacgaggc gtgaagaacc gcggacagat
     1681 tgtacgactt cgaaggcgga tagactgacg gggacggctt agacacacac tagacataga
     1741 cctttctctc gcatagacgg ctcgctgccc ttgttgtagc tcgtacggtt gctctcgtcg
     1801 gaacgaactc gagaatgttg ccctcttagc ctcgcctgat cccgttcggt ccagcgtcgg
     1861 cgacagtgcg aggacatact agactgcttg gaaccgcaag caagcccatc ggagcgactt
     1921 gctgaatgac gacctcatcg ggccgcctcg ctcgtcgctt tcgctctgct ttcactcacc
     1981 acgctacgaa ccatcaatgg gtaccactct tgacgacacg gcttaccggt accgcaccag
     2041 tgtcccgggg gacgccgagg ccatcgaggc actggatggg tccttcacca ccgacaccgt
     2101 cttccgcgtc accgccaccg gggacggctt caccctgcgg gaggtgccga tggacccgcc
     2161 cctgaccaag gtgttccccg acgacgaatc ggacgacgaa tcggacgacg gggaggacgg
     2221 cgacccgggc tcccggacgt tcgtcgcgta cggggacgac ggcgacctgg cgggcttcgt
     2281 ggtcgtcttg tactccggct ggaaccgccg gctgaccgtc gaggacatcg aggtcgcccc
     2341 ggagcaccgg gggcacgggg tcgggcgcgc gttgatgggg ctcgcgacgg agttcgcccg
     2401 cgagcggggc gccgggcacc tctggctgga ggtcaccaac gttaacgcac cggctatcca
     2461 cacgtaccgg cggatggggt tcaccctctg cggcctggac accgccctgt acgacggcac
     2521 cgcctcggac ggcgagcagg cgctctacat gagcatgccc tgcccctaac gttcaaacat
     2581 ttggcaataa agtttcttaa gattgaatcc tgttgccggt cttgcgatga ttatcatata
     2641 atttctgttg aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttat
     2701 gagatgggtt tttatgatta gagtcccgca attatacatt taatacgcga tagaaaacaa
     2761 aatatagcgc gcaaactagg ataaattatc gcgcgcggtg tcatctatgt tactagatcg
     2821 ggacggatgt ggcgaccggg cgcgcgtttc ttgccctcgg cgcaagtggg cattcgctca
     2881 ggcgggcagg gttcttcggc ggtagttcac gcgcatcagt acgtgccgcg ccctttcttc
     2941 gccttactta ttgtgagctg acgcactctt gcacagtaac gtacaaagcg actcggaaga
     3001 cgacgacggc tttgcgcacg tccacgccaa gcacgcctac gagaattttg acgaggttgc
     3061 gccgcacgag attgacccgt acgtgccgct gtctctttcg aatgcatcac aagctcgagc
     3121 tgatgccggc cgcaacaccc agcctccgcg atgttgcggc ttcgttcctc ctcgaaatgt
     3181 tcgaggaact ctggcgaatt atgaagccga acgagtatcg cggcgacaac aaaagcgacc
     3241 tcctcgctca catccaccga cactgcctcc cctcgctcct caagcacata ccgcctcgga
     3301 tccaccacac cgtcgcgccc gaaacggtct acccattcct gtagtcttct taattaagac
     3361 gtcagaattc tcgaggcggc cgcatgtgcg tctccctata gtgagtcgta ttaatttcgc
     3421 gggcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     3481 tagcaccagg cgtttaaggg caccaataac tgccttaaaa aaattacgcc ccgccctgcc
     3541 actcatcgca gtactgttgt aattcattaa gcattctgcc gacatggaag ccatcacaaa
     3601 cggcatgatg aacctgaatc gccagcggca tcagcacctt gtcgccttgc gtataatatt
     3661 tgcccatggt gaaaacgggg gcgaagaagt tgtccatatt ggccacgttt aaatcaaaac
     3721 tggtgaaact cacccaggga ttggctgaga cgaaaaacat attctcaata aaccctttag
     3781 ggaaataggc caggttttca ccgtaacacg ccacgtcttg cgaatatatg tgtagaaact
     3841 gccggaaatc gtcgtggtat tcactccaga gcgatgaaaa cgtttcagtt tgctcatgga
     3901 aaacggtgta acaagggtga acactatccc atatcaccag ctcaccgtct ttcattgcca
     3961 tacggaattc cggatgagca ttcatcaggc gggcaagaat gtgaataaag gccggataaa
     4021 acttgtgctt atttttcttt acggtcttta aaaaggccgt aatatccagc tgaacggtct
     4081 ggttataggt acattgagca actgactgaa atgcctcaaa atgttcttta cgatgccatt
     4141 gggatatatc aacggtggta tatccagtga tttttttctc cattttagct tccttagctc
     4201 ctggaaatct cgataactca aaaaatacgc ccggtagtga tcttatttca ttatggtgaa
     4261 agttggaacc tcttacgtgc cgatcaacgt ctcattttcg ccaaaagttg tcatgaccaa
     4321 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcgaagg
     4381 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     4441 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     4501 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     4561 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     4621 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttgcc
     4681 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     4741 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     4801 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     4861 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     4921 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     4981 cagcaatgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     5041 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     5101 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag ccgaaaaatc
     5161 aataatcaga caacaagatg tgcgaactcg atattttaca cgactctctt taccaattct
     5221 gccccgaatt acacttaaaa cgactcaaca gcttaacgtt ggcttgccac gcattacttg
     5281 actgtaaaac tctcactctt accgaacttg gccgtaacct gccaaccaaa gcgagaacaa
     5341 aacataacat caaacgaatc gaccgattgt taggtaatcg tcacctgcag gaaggtttaa
     5401 acgcatttag gtgacactat agaagtgtgt atcgctcgag ggatccgaat tcgaa
//
