LOCUS       sequence-386128-        4778 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  integration cassette for ku70 deletion and expression of NAT gene 
            under pGPD for characterization.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4778)
  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 4778)
  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+pGPD+NAT+tNOS+ku70 insD (SBE175)
FEATURES             Location/Qualifiers
     source          1..4778
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1315..1890
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVISYSAWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFAGERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGERQALYMSMPCP"
     primer_bind     complement(2717..2736)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2718..2736)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(2847..3506)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3421..3440
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(3507..3609)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      3710..4298
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4199..4218
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4452..4469
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4728..4746
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4728..4745
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gacttggtac ggagccgcct cctccacctc agcaaccaac ctaccagtcg caccccggat
       61 cgcagtctag tcttccgccg cttcccgctc tcccttcttc ctcgatgccg cctcctccgc
      121 ttcctcagcc caaggcgcct cagagcgccg catcctccgc ctcgacgctt ccgcctccgc
      181 cattctaggc gatgccggga ggaaccatgc 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 cggtgtgact gatctggtgt
      541 tgttctgagc gggtagagcg agagcgggtg atgggaaaga gggcaaaggg acgcgaactt
      601 gtcgatgcgg cttccttcgc tcacgaaatc agccctcgcg cgtttcgtcc cccggctgga
      661 cccgacctgc gctcgaggcg ggcgtgtgac ggcttcctgc gctgcagatt cagacggatc
      721 atgctctcga gggattgttt ccctccttca ccgcttcgct cgccctctct gcgacgtcgg
      781 ctgagatcga taagttggtg atgtgacgtg gccatctccg aggttccttt ccgaccctta
      841 caagccgaga ttcgcgcagc ttcgattgat tgtattgctc gaggtaaccg agtatgaggg
      901 agtcgtctct cgtcgacccc aagcctgcgc atactgctgc ccgcttctgg tgcctgctcc
      961 aataaattac tgtacttggc gccgcgattt ctggactatc tcgcttgcct tatgctcccg
     1021 cggcgccgct ctgcgccgtt cctcccacga cttccatgtc ctcacagctt gtttcgtcgt
     1081 ccgaagcgtc ctctacgtct tgcaaacgtc aaaaaacgac gttggcatcc cggcggcaca
     1141 aggcttgctc cggtaggcga cctacaaggc gagccagcag atttgcttgt ccgcagagat
     1201 ttcttccttc catcgtgccg tctaaaggac gaaacgaggt cacggaggag ggaactagag
     1261 agcaaggttc attccaatgc ttgcggagcg aggagaacgt cgaactccat atcaatgggt
     1321 accactcttg acgacacggc ttaccggtac cgcaccagtg tcccggggga cgccgaggcc
     1381 atcgaggcac tggatgggtc cttcaccacc gacaccgtct tccgcgtcac cgccaccggg
     1441 gacggcttca ccctgcggga ggtgccggtg gacccgcccc tgaccaaggt gttccccgac
     1501 gacgaatcgg acgacgaatc ggacgacggg gaggacggcg acccggactc ccggacgttc
     1561 gtcgcgtacg gggacgacgg cgacctggcg ggcttcgtgg tcatctcgta ctcggcgtgg
     1621 aaccgccggc tgaccgtcga ggacatcgag gtcgccccgg agcaccgggg gcacggggtc
     1681 gggcgcgcgt tgatggggct cgcgacggag ttcgccggcg agcggggcgc cgggcacctc
     1741 tggctggagg tcaccaacgt caacgcaccg gcgatccacg cgtaccggcg gatggggttc
     1801 accctctgcg gcctggacac cgccctgtac gacggcaccg cctcggacgg cgagcggcag
     1861 gcgctctaca tgagcatgcc ctgcccctaa tccgttcaaa catttggcaa taaagtttct
     1921 taagattgaa tcctgttgcc ggtcttgcga tgattatcat ataatttctg ttgaattacg
     1981 ttaagcatgt aataattaac atgtaatgca tgacgttatt tatgagatgg gtttttatga
     2041 ttagagtccc gcaattatac atttaatacg cgatagaaaa caaaatatag cgcgcaaact
     2101 aggataaatt atcgcgcgcg gtgtcatcta tgttactaga tcggggtgta tgtggcgacc
     2161 gggcgcgcgt ttcttgccct cggcgcaagt gggcattcgc tcaggcgggc agggttcttc
     2221 ggcggtagtt cacgcgcatc agtacgtgcc gcgccctttc ttcgccttac ttattgtgag
     2281 ctgacgcact cttgcacagt aacgtacaaa gcgactcgga agacgacgac ggctttgcgc
     2341 acgtccacgc caagcacgcc tacgagaatt ttgacgaggt tgcgccgcac gagattgacc
     2401 cgtacgtgcc gctgtctctt tcgaatgcat cacaagctcg agctgatgcc ggccgcaaca
     2461 cccagcctcc gcgatgttgc ggcttcgttc ctcctcgaaa tgttcgagga actctggcga
     2521 attatgaagc cgaacgagta tcgcggcgac aacaaaagcg acctcctcgc tcacatccac
     2581 cgacactgcc tcccctcgct cctcaagcac atcccgcctc ggatccacca caccgtcgcg
     2641 cccgaaacgg tctacccatt cctgtagtct tcttaattaa gacgtcagaa ttctcgaggc
     2701 ggccgcatgt gcgtctccct atagtgagtc gtattaattt cgcgggcgga acccctattt
     2761 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagtagcacc aggcgtttaa
     2821 gggcaccaat aactgcctta aaaaaattac gccccgccct gccactcatc gcagtactgt
     2881 tgtaattcat taagcattct gccgacatgg aagccatcac aaacggcatg atgaacctga
     2941 atcgccagcg gcatcagcac cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg
     3001 ggggcgaaga agttgtccat attggccacg tttaaatcaa aactggtgaa actcacccag
     3061 ggattggctg agacgaaaaa catattctca ataaaccctt tagggaaata ggccaggttt
     3121 tcaccgtaac acgccacatc ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg
     3181 tattcactcc agagcgatga aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg
     3241 tgaacactat cccatatcac cagctcaccg tctttcattg ccatacggaa ttccggatga
     3301 gcattcatca ggcgggcaag aatgtgaata aaggccggat aaaacttgtg cttatttttc
     3361 tttacggtct ttaaaaaggc cgtaatatcc agctgaacgg tctggttata ggtacattga
     3421 gcaactgact gaaatgcctc aaaatgttct ttacgatgcc attgggatat atcaacggtg
     3481 gtatatccag tgattttttt ctccatttta gcttccttag ctcctgaaaa tctcgataac
     3541 tcaaaaaata cgcccggtag tgatcttatt tcattatggt gaaagttgga acctcttacg
     3601 tgccgatcaa cgtctcattt tcgccaaaag ttgtcatgac caaaatccct taacgtgagt
     3661 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     3721 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     3781 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     3841 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     3901 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     3961 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     4021 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     4081 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     4141 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     4201 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     4261 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaat gcggcctttt
     4321 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     4381 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     4441 cgaccgagcg cagcgagtca gtgagcgagg aagccgaaaa atcaataatc agacaacaag
     4501 atgtgcgaac tcgatatttt acacgactct ctttaccaat tctgccccga attacactta
     4561 aaacgactca acagcttaac gttggcttgc cacgcattac ttgactgtaa aactctcact
     4621 cttaccgaac ttggccgtaa cctgccaacc aaagcgagaa caaaacataa catcaaacga
     4681 atcgaccgat tgttaggtaa tcgtcacctg caggaaggtt taaacgcatt taggtgacac
     4741 tatagaagtg tgtatcgctc gagggatccg aattcgaa
//
