LOCUS       sequence-386093-        4495 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  the insertional region downstream for the deletion of the KU70 gene 
            in R. toruloides. Overhangs S/Q.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4495)
  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 4495)
  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: (S) insD_ku70 (Q) (SBE139)
FEATURES             Location/Qualifiers
     source          1..4495
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        242..260
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(867..886)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(868..886)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(893..910)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(893..909)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(902..924)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1047..1349
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1228..1247
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1698..2492
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             1698..2492
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1752..1771)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2362..2381
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             complement(join(2742..3533,3534..3602))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3365..3384
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3726..4314
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4215..4234
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4468..4485
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctca
      241 gaattaaccc tcactaaagg gactagtcct gcaggtttaa acgaattcgc ccttagagca
      301 ggtctcctcg ggatgtggcg accgggcgcg cgtttcttgc cctcggcgca agtgggcatt
      361 cgctcaggcg ggcagggttc ttcggcggta gttcacgcgc atcagtacgt gccgcgccct
      421 ttcttcgcct tacttattgt gagctgacgc actcttgcac agtaacgtac aaagcgactc
      481 ggaagacgac gacggctttg cgcacgtcca cgccaagcac gcctacgaga attttgacga
      541 ggttgcgccg cacgagattg acccgtacgt gccgctgtct ctttcgaatg catcacaagc
      601 tcgagctgat gccggccgca acacccagcc tccgcgatgt tgcggcttcg ttcctcctcg
      661 aaatgttcga ggaactctgg cgaattatga agccgaacga gtatcgcggc gacaacaaaa
      721 gcgacctcct cgctcacatc caccgacact gcctcccctc gctcctcaag cacatcccgc
      781 ctcggatcca ccacaccgtc gcgcccgaaa cggtctaccc attgagaccg cataagggcg
      841 aattcgcggc cgctaaattc aattcgccct atagtgagtc gtattacaat tcactggccg
      901 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
      961 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     1021 aacagttgcg cagcctatac gtacggcagt ttaaggttta cacctataaa agagagagcc
     1081 gttatcgtct gtttgtggat gtacagagtg atattattga cacgccgggg cgacggatgg
     1141 tgatccccct ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa ctttacccgg
     1201 tggtgcatat cggggatgaa agctggcgca tgatgaccac cgatatggcc agtgtgccgg
     1261 tctccgttat cggggaagaa gtggctgatc tcagccaccg cgaaaatgac atcaaaaacg
     1321 ccattaacct gatgttctgg ggaatataaa tgtcaggcat gagattatca aaaaggatct
     1381 tcacctagat ccttttcacg tagaaagcca gtccgcagaa acggtgctga ccccggatga
     1441 atgtcagcta ctgggctatc tggacaaggg aaaacgcaag cgcaaagaga aagcaggtag
     1501 cttgcagtgg gcttacatgg cgatagctag actgggcggt tttatggaca gcaagcgaac
     1561 cggaattgcc agctggggcg ccctctggta aggttgggaa gccctgcaaa gtaaactgga
     1621 tggctttctt gccgccaagg atctgatggc gcaggggatc aagctctgat caagagacag
     1681 gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
     1741 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     1801 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     1861 gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc gtggctggcc acgacgggcg
     1921 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     1981 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
     2041 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
     2101 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
     2161 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
     2221 aggcgagcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga
     2281 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
     2341 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
     2401 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
     2461 ccttctatcg ccttcttgac gagttcttct gaattattaa cgcttacaat ttcctgatgc
     2521 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catcaggtgg cacttttcgg
     2581 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     2641 ctcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     2701 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     2761 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     2821 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     2881 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     2941 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     3001 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     3061 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     3121 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     3181 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     3241 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     3301 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     3361 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     3421 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     3481 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     3541 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     3601 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgaccaaa
     3661 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     3721 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     3781 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     3841 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     3901 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     3961 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     4021 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     4081 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     4141 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     4201 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     4261 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     4321 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     4381 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     4441 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaag
//
