LOCUS       sequence-386129-        4753 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  integration cassette for ku70 deletion and expression of NAT gene 
            under pFBA for characterization.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4753)
  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 4753)
  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+pFBA+NAT+tNOS+ku70 insD (SBE179)
FEATURES             Location/Qualifiers
     source          1..4753
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1296..1868
                     /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(2692..2711)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2693..2711)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(2822..3481)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3396..3415
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(3482..3584)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      3685..4273
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4174..4193
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4427..4444
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4703..4721
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4703..4720
                     /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 ggttccgatt
      541 ctctgatcca gaagcacgcc ggctcgaagc tgttcggcaa ctttgtcgac gaactcgcgc
      601 gcctgcttgc cgcgccgctc aagtcggacg aggtgggcaa ggtgcgcgcg agtatggcca
      661 acctcgcgat ggacaagcag aagctcgaga aggctggtac gaagggcggt gcggtcggtg
      721 ggaagccccc tgctaggatg gtcgcgaggg gaagggagga cttgtcgtcg ttcggggagg
      781 tgctcgacga cgatgtcgcc gcggcccaat tcgacgagga cgaggatttc gtgcgtcgct
      841 ttcgctcgct cgctggctgc tcctgtctct tctgcttctc acgctgactc tcatcgtgtt
      901 cgtctccccg cagatgtaga tgtagacgcg cctttttcca gcttcacccg cttccaaccc
      961 ttccacctgc aaccgcactt tcgccccgtt cctccgctct cttgcggctg cgatgttgtc
     1021 cagcgtcgac aggagctgct tcactctcgc ctgacctgct tgccacctgc tgctcgcacg
     1081 atgccatcgc gaggtgggcg agagagcgtg gctggatgac gctcgctccg gcttgcagct
     1141 ggttgttacg gtgttgcaag aatttctgta cagtttgtac gagtggcccc gcgttgtgga
     1201 tgatgtcggt tcggttggcg cggccttgct cgctcgctct ctcgttgctc ctcgctcttc
     1261 accacttcac ttctaacact aactagctac aatcaatggg taccactctt gacgacacgg
     1321 cttaccggta ccgcaccagt gtcccggggg acgccgaggc catcgaggca ctggatgggt
     1381 ccttcaccac cgacaccgtc ttccgcgtca ccgccaccgg ggacggcttc accctgcggg
     1441 aggtgccgat ggacccgccc ctgaccaagg tgttccccga cgacgaatcg gacgacgaat
     1501 cggacgacgg ggaggacggc gacccgggct cccggacgtt cgtcgcgtac ggggacgacg
     1561 gcgacctggc gggcttcgtg gtcgtcttgt actccggctg gaaccgccgg ctgaccgtcg
     1621 aggacatcga ggtcgccccg gagcaccggg ggcacggggt cgggcgcgcg ttgatggggc
     1681 tcgcgacgga gttcgcccgc gagcggggcg ccgggcacct ctggctggag gtcaccaacg
     1741 ttaacgcacc ggctatccac acgtaccggc ggatggggtt caccctctgc ggcctggaca
     1801 ccgccctgta cgacggcacc gcctcggacg gcgagcaggc gctctacatg agcatgccct
     1861 gcccctaacg ttcaaacatt tggcaataaa gtttcttaag attgaatcct gttgccggtc
     1921 ttgcgatgat tatcatataa tttctgttga attacgttaa gcatgtaata attaacatgt
     1981 aatgcatgac gttatttatg agatgggttt ttatgattag agtcccgcaa ttatacattt
     2041 aatacgcgat agaaaacaaa atatagcgcg caaactagga taaattatcg cgcgcggtgt
     2101 catctatgtt actagatcgg gacggatgtg gcgaccgggc gcgcgtttct tgccctcggc
     2161 gcaagtgggc attcgctcag gcgggcaggg ttcttcggcg gtagttcacg cgcatcagta
     2221 cgtgccgcgc cctttcttcg ccttacttat tgtgagctga cgcactcttg cacagtaacg
     2281 tacaaagcga ctcggaagac gacgacggct ttgcgcacgt ccacgccaag cacgcctacg
     2341 agaattttga cgaggttgcg ccgcacgaga ttgacccgta cgtgccgctg tctctttcga
     2401 atgcatcaca agctcgagct gatgccggcc gcaacaccca gcctccgcga tgttgcggct
     2461 tcgttcctcc acgaaatgtt cgaggaactc tggcgaatta tgaagccgaa cgagtatcgc
     2521 ggcgacaaca aaagcgacct cctcgctcac atccaccgac actgcctccc ctcgctcctc
     2581 aagcacatcc cgcctcggat ccaccacacc gtcgcgccga aacggtctac ccattcctgt
     2641 agtcttctta attaagacgt cagaattctc gaggcggccg catgtgcgtc tccctatagt
     2701 gagtcgtatt aatttcgcgg gcggaacccc tatttgttta tttttctaaa tacattcaaa
     2761 tatgtatccg ctcatgagta gcaccaggcg tttaagggca ccaataactg ccttaaaaaa
     2821 attacgcccc gccctgccac tcatcgcagt actgttgtaa ttcattaagc attctgccga
     2881 catggaagcc atcacaaacg gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt
     2941 cgccttgcgt ataatatttg cccatggtga aaacgggggc gaagaagttg tccatattgg
     3001 ccacgtttaa atcaaaactg gtgaaactca cccagggatt ggctgagacg aaaaacatat
     3061 tctcaataaa ccctttaggg aaataggcca ggttttcacc gtaacacgcc acgtcttgcg
     3121 aatatatgtg tagaaactgc cggaaatcgt cgtggtattc actccagagc gatgaaaacg
     3181 tttcagtttg ctcatggaaa acggtgtaac aagggtgaac actatcccat atcaccagct
     3241 caccgtcttt cattgccata cggaattccg gatgagcatt catcaggcgg gcaagaatgt
     3301 gaataaaggc cggataaaac ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa
     3361 tatccagctg aacggtctgg ttataggtac attgagcaac tgactgaaat gcctcaaaat
     3421 gttctttacg atgccattgg gatatatcaa cggtggtata tccagtgatt tttttctcca
     3481 ttttagcttc cttagctcct ggaaatctcg ataactcaaa aaatacgccc ggtagtgatc
     3541 ttatttcatt atggtgaaag ttggaacctc ttacgtgccg atcaacgtct cattttcgcc
     3601 aaaagttgtc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     3661 cgtagaaaag atcgaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     3721 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     3781 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     3841 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     3901 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     3961 ctcaagacga tagttgccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     4021 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     4081 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     4141 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     4201 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     4261 gagcctatgg aaaaacgcca gcaatgcggc ctttttacgg ttcctggcct tttgctggcc
     4321 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     4381 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     4441 cgaggaagcc gaaaaatcaa taatcagaca acaagatgtg cgaactcgat attttacacg
     4501 actctcttta ccaattctgc cccgaattac acttaaaacg actcaacagc ttaacgttgg
     4561 cttgccacgc attacttgac tgtaaaactc tcactcttac cgaacttggc cgtaacctgc
     4621 caaccaaagc gagaacaaaa cataacatca aacgaatcga ccgattgtta ggtaatcgtc
     4681 acctgcagga aggtttaaac gcatttaggt gacactatag aagtgtgtat cgctcgaggg
     4741 atccgaattc gaa
//
