LOCUS       sequence-386130-        4746 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  integration cassette for ku70 deletion and expression of NAT gene 
            under pADH2 for characterization.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4746)
  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 4746)
  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+pADH2+NAT+tNOS+ku70 insD (SBE174)
FEATURES             Location/Qualifiers
     source          1..4746
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1289..1861
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDGTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPMDPPLTKVFPDDESDDESDDGEDGDPGSRTFVAYGDDGDLAGFVVVLYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHTYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     primer_bind     complement(2685..2704)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2686..2704)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(2815..3474)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3389..3408
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(3475..3577)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      3678..4266
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4167..4186
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4420..4437
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4696..4714
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4696..4713
                     /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 ggttttgcag
      541 ttacagttcg ttaaatatcg gctgaggctt ccccgacgcc cctcctcccc tccctcctcg
      601 ccctcctcct cgtcctcctc gtgatgccat cagtcgcgcc ggcggggtgc tgcggtttgc
      661 gacggtgttc tttgagggtg tgtgtgcgct gagcaagcag tagcaggcag tccagcaggt
      721 gagcgcggtg tgggaacagc tcgagcttgt tgcgggcggt gtactgatca agtaggacct
      781 cggagactac ggtcagagcc tacgacgacg tcgttgcagt cgtgtacact gggaatggca
      841 gctaggctga tccccgccgc tctccaccac actttactcg tcttccgtcc tcgcccgcag
      901 tcgttaattc atgggtgggg acgtcgaagg gtgggctttg cttcggggtg gctgggtccc
      961 gtgctcgagt ggctgtgggc gtcaattaag cgtgtctcgt gcgagagcga gccgacttgc
     1021 gagtcgtccc gagcaggagt ggccttcctc gctcttcgcc ttccccaccc atcgaccgtc
     1081 ccgcaacctt gcgctcgctc tctctcttac agcttgctct tcacttgcac acactgctac
     1141 acaccctcgc acagcactcg cactcagccc ataccggtcc agccttcgca gccctctctc
     1201 ttcgtgcgtt cccattcccg gctgttcact ctcgcaccgt catttcccgt ctagctgaca
     1261 gctgccacgt ctccgacagt cacaatcaat gggtaccact cttgacggca cggcttaccg
     1321 gtaccgcacc agtgtcccgg gggacgccga ggccatcgag gcactggatg ggtccttcac
     1381 caccgacacc gtcttccgcg tcaccgccac cggggacggc ttcaccctgc gggaggtgcc
     1441 gatggacccg cccctgacca aggtgttccc cgacgacgaa tcggacgacg aatcggacga
     1501 cggggaggac ggcgacccgg gctcccggac gttcgtcgcg tacggggacg acggcgacct
     1561 ggcgggcttc gtggtcgtct tgtactccgg ctggaaccgc cggctgaccg tcgaggacat
     1621 cgaggtcgcc ccggagcacc gggggcacgg ggtcgggcgc gcgttgatgg ggctcgcgac
     1681 ggagttcgcc cgcgagcggg gcgccgggca cctctggctg gaggtcacca acgttaacgc
     1741 accggctatc cacacgtacc ggcggatggg gttcaccctc tgcggcctgg acaccgccct
     1801 gtacgacggc accgcctcgg acggcgagca ggcgctctac atgagcatgc cctgccccta
     1861 acgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg gtcttgcgat
     1921 gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca tgtaatgcat
     1981 gacgttattt atgagatggg tttttatgat tagagtcccg caattataca tttaatacgc
     2041 gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg tgtcatctat
     2101 gttactagat cgggacggat gtggcgaccg ggcgcgcgtt tcttgccctc ggcgcaagtg
     2161 ggcattcgct caggcgggca gggttcttcg gcggtagttc acgcgcatca gtacgtgccg
     2221 cgccctttct tcgccttact tattgtgagc tgacgcactc ttgcacagta acgtacaaag
     2281 cgactcggaa gacgacgacg gctttgcgca cgtccacgcc aagcacgcct acgagaattt
     2341 tgacgaggtt gcgccgcacg agattgaccc gtacgtgccg ctgtctcttt cgaatgcatc
     2401 acaagctcga gctgatgccg gccgcaacac ccagcctccg cgatgttgcg gcttcgttcc
     2461 tccacgaaat gttcgaggaa ctctggcgaa ttatgaagcc gaacgagtat cgcggcgaca
     2521 acaaaagcga cctcctcgct cacatccacc gacactgcct cccctcgctc ctcaagcaca
     2581 tcccgcctcg gatccaccac accgtcgcgc cgaaacggtc tacccattcc tgtagtcttc
     2641 ttaattaaga cgtcagaatt ctcgaggcgg ccgcatgtgc gtctccctat agtgagtcgt
     2701 attaatttcg cgggcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     2761 ccgctcatga gtagcaccag gcgtttaagg gcaccaataa ctgccttaaa aaaattacgc
     2821 cccgccctgc cactcatcgc agtactgttg taattcatta agcattctgc cgacatggaa
     2881 gccatcacaa acggcatgat gaacctgaat cgccagcggc atcagcacct tgtcgccttg
     2941 cgtataatat ttgcccatgg tgaaaacggg ggcgaagaag ttgtccatat tggccacgtt
     3001 taaatcaaaa ctggtgaaac tcacccaggg attggctgag acgaaaaaca tattctcaat
     3061 aaacccttta gggaaatagg ccaggttttc accgtaacac gccacgtctt gcgaatatat
     3121 gtgtagaaac tgccggaaat cgtcgtggta ttcactccag agcgatgaaa acgtttcagt
     3181 ttgctcatgg aaaacggtgt aacaagggtg aacactatcc catatcacca gctcaccgtc
     3241 tttcattgcc atacggaatt ccggatgagc attcatcagg cgggcaagaa tgtgaataaa
     3301 ggccggataa aacttgtgct tatttttctt tacggtcttt aaaaaggccg taatatccag
     3361 ctgaacggtc tggttatagg tacattgagc aactgactga aatgcctcaa aatgttcttt
     3421 acgatgccat tgggatatat caacggtggt atatccagtg atttttttct ccattttagc
     3481 ttccttagct cctggaaatc tcgataactc aaaaaatacg cccggtagtg atcttatttc
     3541 attatggtga aagttggaac ctcttacgtg ccgatcaacg tctcattttc gccaaaagtt
     3601 gtcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     3661 aagatcgaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     3721 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     3781 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     3841 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     3901 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     3961 cgatagttgc cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     4021 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     4081 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     4141 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     4201 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     4261 tggaaaaacg ccagcaatgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     4321 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     4381 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     4441 gccgaaaaat caataatcag acaacaagat gtgcgaactc gatattttac acgactctct
     4501 ttaccaattc tgccccgaat tacacttaaa acgactcaac agcttaacgt tggcttgcca
     4561 cgcattactt gactgtaaaa ctctcactct taccgaactt ggccgtaacc tgccaaccaa
     4621 agcgagaaca aaacataaca tcaaacgaat cgaccgattg ttaggtaatc gtcacctgca
     4681 ggaaggttta aacgcattta ggtgacacta tagaagtgtg tatcgctcga gggatccgaa
     4741 ttcgaa
//
