LOCUS       sequence-386081-        4500 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  the insertional region upstream for the deletion of the KU70 gene in
            R. toruloides. Overhangs P/B.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4500)
  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 4500)
  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: (P) insUP_ku70 (SBE138) (B)
FEATURES             Location/Qualifiers
     source          1..4500
                     /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(872..891)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(873..891)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(898..915)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(898..914)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(907..929)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1052..1354
                     /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     1233..1252
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1703..2497
                     /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             1703..2497
                     /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(1757..1776)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2367..2386
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             complement(join(2747..3538,3539..3607))
                     /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     3370..3389
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3731..4319
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4220..4239
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4473..4490
                     /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 ccttgcatgg
      301 tctcaggagc cgcctcctcc acctcagcaa ccaacctacc agtcgcaccc cggatcgcag
      361 tctagtcttc cgccgcttcc cgctctccct tcttcctcga tgccgcctcc tccgcttcct
      421 cggcccaagg cgcctcagag cgccgcatcc tccgcctcaa cgcttccgcc tccgccattc
      481 taggcgatgc cgggaggaac cgtgctgaac ggacaaggta cggccccggc ggcagtccag
      541 gcgaacgggg tgcagcagtg aggagcgaac agtatgcata gctggatcgt cgcatgcgtc
      601 ttagtgtctt gctttcttgt tggaaaatcg ttggaatcgt tggaccttgt tgtagcctct
      661 ctcacgcaac gactggcttg tttgacgtct cttcattgcg tggggagagt gaagcgatgt
      721 ttgcaaagta ggacacaaca agcgaacgga ctactgcgaa cgtacacagg cgagagcgat
      781 cgggggcgaa agtcagtcgt cgtcctccca tcgccgacga tgaaggttga gaccgcataa
      841 gggcgaattc gcggccgcta aattcaattc gccctatagt gagtcgtatt acaattcact
      901 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
      961 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     1021 ttcccaacag ttgcgcagcc tatacgtacg gcagtttaag gtttacacct ataaaagaga
     1081 gagccgttat cgtctgtttg tggatgtaca gagtgatatt attgacacgc cggggcgacg
     1141 gatggtgatc cccctggcca gtgcacgtct gctgtcagat aaagtctccc gtgaacttta
     1201 cccggtggtg catatcgggg atgaaagctg gcgcatgatg accaccgata tggccagtgt
     1261 gccggtctcc gttatcgggg aagaagtggc tgatctcagc caccgcgaaa atgacatcaa
     1321 aaacgccatt aacctgatgt tctggggaat ataaatgtca ggcatgagat tatcaaaaag
     1381 gatcttcacc tagatccttt tcacgtagaa agccagtccg cagaaacggt gctgaccccg
     1441 gatgaatgtc agctactggg ctatctggac aagggaaaac gcaagcgcaa agagaaagca
     1501 ggtagcttgc agtgggctta catggcgata gctagactgg gcggttttat ggacagcaag
     1561 cgaaccggaa ttgccagctg gggcgccctc tggtaaggtt gggaagccct gcaaagtaaa
     1621 ctggatggct ttcttgccgc caaggatctg atggcgcagg ggatcaagct ctgatcaaga
     1681 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     1741 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     1801 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     1861 gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac
     1921 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     1981 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     2041 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     2101 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     2161 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     2221 gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     2281 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     2341 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     2401 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     2461 catcgccttc tatcgccttc ttgacgagtt cttctgaatt attaacgctt acaatttcct
     2521 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatca ggtggcactt
     2581 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     2641 atccgctcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     2701 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     2761 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     2821 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     2881 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     2941 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     3001 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     3061 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     3121 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     3181 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     3241 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     3301 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     3361 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     3421 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     3481 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     3541 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     3601 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     3661 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     3721 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     3781 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     3841 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     3901 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     3961 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     4021 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     4081 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     4141 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     4201 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     4261 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     4321 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     4381 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     4441 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
//
