LOCUS       sequence-338229-        8509 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  constitutive expression of TIR1-myc and tTA in sleeping beauty 
            transposon vector with selection marker (G418).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8509)
  AUTHORS   Yeung TK, Lau HW, Ma HT, Poon RYC
  TITLE     One-step multiplex toolkit for efficient generation of conditional 
            gene silencing human cell lines.
  JOURNAL   Mol Biol Cell. 2021 May 12:mbcE21020051. doi: 
            10.1091/mbc.E21-02-0051.
  PUBMED    33979199
REFERENCE   2  (bases 1 to 8509)
  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: "Mol Biol 
            Cell. 2021 May 12:mbcE21020051. doi: 10.1091/mbc.E21-02-0051."
COMMENT     Sequence Label: pSBbi-TIR1-tTA/Neo
FEATURES             Location/Qualifiers
     source          1..8509
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     58..77
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(275..294)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    340..395
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(394..413)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(787..804)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(958..1546)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1038..1057)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1717..2508,2509..2577))
                     /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     2340..2359
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2578..2682)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    3114..3338
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3327..3344
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(3401..3430)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             complement(3437..3466)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             complement(3473..3502)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             complement(3530..5254)
                     /codon_start=1
                     /product="rice transport inhibitor response 1 protein"
                     /label=OsTIR1
                     /note="F-box protein that mediates auxin-dependent 
                     interaction of a ubiquitin ligase with an auxin-inducible 
                     degron (Tan et al., 2007)"
                     /translation="MAYFPEEVVEHIFSFLPAQRDRNTVSLVCKVWYEIERLSRRGVFV
                     GNCYAVRAGRVAARFPNVRALTVKGKPHFADFNLVPPDWGGYAGPWIEAAARGCHGLEE
                     LRMKRMVVSDESLELLARSFPRFRALVLISCEGFSTDGLAAVASHCKLLRELDLQENEV
                     EDRGPRWLSCFPDSCTSLVSLNFACIKGEVNAGSLERLVSRSPNLRSLRLNRSVSVDTL
                     AKILLRTPNLEDLGTGNLTDDFQTESYFKLTSALEKCKMLRSLSGFWDASPVCLSFIYP
                     LCAQLTGLNLSYAPTLDASDLTKMISRCVKLQRLWVLDCISDKGLQVVASSCKDLQELR
                     VFPSDFYVAGYSAVTEEGLVAVSLGCPKLNSLLYFCHQMTNAALVTVAKNCPNFTRFRL
                     CILEPGKPDVVTSQPLDEGFGAIVRECKGLQRLSISGLLTDKVFMYIGKYAKQLEMLSI
                     AFAGDSDKGMMHVMNGCKNLRKLEIRDSPFGDAALLGNFARYETMRSLWMSSCNVTLKG
                     CQVLASKMPMLNVEVINERDGSNEMEENHGDLPKVEKLYVYRTTAGARDDAPNFVKIL"
     regulatory      5251..5260
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5286..6468)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     primer_bind     complement(5318..5338)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     complement(7169..7188)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             7723..7833
                     /codon_start=1
                     /product="3 tandem repeats of the minimal activation domain
                     of herpes simplex virus VP16 (Cheng et al., 2013)"
                     /label=VP48
                     /translation="DALDDFDLDMLPADALDDFDLDMLPADALDDFDLDML"
     CDS             7846..7899
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(7900..8509,1..188)
                     /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="SMIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
                     PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLL
                     SSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEH
                     QGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDI
                     ALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(7957..7976)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
       61 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
      121 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
      181 tcttctgagc ggaattcact cctcaggtgc aggctgccta tcagaaggtg gtggctggtg
      241 tggccaatgc cctggctcac aaataccact gagatctttt tccctctgcc aaaaattatg
      301 gggacatcat gaagcccctt gagcatctga cttctggcta ataaaggaaa tttattttca
      361 ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg aaggacatat gggagggcaa
      421 atcatttaaa acatcagaat gagtatttgg tttagagttt ggcaacatat gccatatgct
      481 ggctgccatg aactagctac tcgggacccc ttaccgaaac atcgccgcat tctgcagagg
      541 agtcgagtgt atgtaaactt ctgacccact gggaatgtga tgaaagaaat aaaagctgaa
      601 atgaatcatt ctctctacta ttattctgat atttcacatt cttaaaataa agtggtgatc
      661 ctaactgacc taagacaggg aatttttact aggattaaat gtcaggaatt gtgaaaaagt
      721 gagtttaaat gtatttggct aaggtgtatg taaacttccg acttcaactg tatagggatc
      781 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
      841 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
      901 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
      961 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     1021 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     1081 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     1141 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     1201 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     1261 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     1321 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     1381 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     1441 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     1501 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     1561 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     1621 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     1681 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     1741 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     1801 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     1861 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     1921 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     1981 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     2041 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     2101 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     2161 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     2221 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     2281 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     2341 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     2401 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     2461 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     2521 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     2581 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     2641 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     2701 gccacctgat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcagga
     2761 aattgtaagc gttaatattt tgttaaaatt ggatccctat acagttgaag tcggaagttt
     2821 acatacactt aagttggagt cattaaaact cgtttttcaa ctactccaca aatttcttgt
     2881 taacaaacaa tagttttggc aagtcagtta ggacatctac tttgtgcatg acacaagtca
     2941 tttttccaac aattgtttac agacagatta tttcacttat aattcactgt atcacaattc
     3001 cagtgggtca gaagtttaca tacactaagt tcgactcctc tgcagaatgc ggcgatgttt
     3061 cggtaagggg tccgctatct agtaggccca gctggttctt tccgcctcag aagccataga
     3121 gcccaccgca tccccagcat gcctgctatt gtcttcccaa tcctccccct tgctgtcctg
     3181 ccccacccca ccccccagaa tagaatgaca cctactcaga caatgcgatg caatttcctc
     3241 attttattag gaaaggacag tgggagtggc accttccagg gtcaaggaag gcacggggga
     3301 ggggcaaaca acagatggct ggcaactaga aggcacagtc gaggctgatc agcgagctct
     3361 agagaattga tccccaagct ttttagctag tggatccgtt caagtcttct tctgagatta
     3421 atttttgttc accgttcaag tcttcctcgg agattagctt ttgttcaccg ttcaaatctt
     3481 cttcagaaat caacttttgt tcaccactag cagcagaacc ggagtcgact aggattttaa
     3541 caaaatttgg tgcatcatcc ctcgccccag cagttgtgcg gtacacatat aatttctcca
     3601 cctttggcag gtcgccatga ttttcctcca tttcgttaga cccgtcccgc tcattaatga
     3661 cttccacgtt cagcataggc attttactag ccaggacctg acagcccttc agagtcacat
     3721 tgcaactaga catccacagg ctcctcattg tctcgtatct ggcaaagttt cccagcagag
     3781 cggcatcccc gaaagggctg tcccggattt ccagttttcg cagattctta cacccgttca
     3841 tcacgtgcat catgccctta tcggagtctc cggcaaatgc aatgctcagc atctccagct
     3901 gcttagcata cttgccgatg tacatgaaca ctttgtctgt cagcaggccg gaaatgctca
     3961 gcctctggag tcccttgcac tctctcacga tagctccaaa cccttcatcc agtggctggg
     4021 atgtgaccac gtcgggtttg cctggttcca gaatacacag ccgaaatcgg gtgaaattag
     4081 ggcagttctt ggcgacggtc accagtgcag cattagtcat ctgatgacag aaatacagca
     4141 ggctgttcag ctttgggcat cccagtgaga ctgccaccag gccttcctca gtgacagcac
     4201 tatatccggc cacgtaaaaa tcagatggga acactctcag ctcctggagg tccttgcagg
     4261 agctggcgac cacctggagc cccttatcgg aaatacagtc cagcacccac agtcgctgga
     4321 gtttcacgca gcgtgagatc atctttgtca gatcactggc gtccagggtt ggtgcatagc
     4381 tcaggttcag gcctgtcagc tgtgcacaca gagggtagat aaagctcagg cacacgggtg
     4441 aagcatccca gaatcctgac agactcctca gcattttaca cttctccagg gcagaagtca
     4501 gtttaaagta ggattctgtc tggaaatcgt ctgtcagatt gccggttccc agatcctcca
     4561 ggttaggggt tctcagcaga atcttagcca gagtgtccac gctcacgctc cggttcagcc
     4621 gcagagacct caggttgggg cttcttgaga ccagccgctc cagggaccca gcattcactt
     4681 cgcctttgat gcaggcaaag ttcagggaca ccaggctggt acatgagtct gggaagcaag
     4741 acagccatct ggggcctcta tcctccactt cattctcctg gaggtccagc tcccgcagca
     4801 gcttacagtg ggaggccacg gctgccagtc catcggtgct gaagccttcg caagaaatca
     4861 gcaccagtgc gcgaaaccta gggaagctcc gagccagcag ctccagactt tcgtcagaga
     4921 ccaccattcg cttcatgcgc agttcctcca gtccatgaca gccgcgagcg gctgcctcga
     4981 tccaagggcc ggcgtatcct ccccaatcag ggggcaccag gttaaagtcg gcgaagtggg
     5041 gtttcccctt gacggtcagt gcgcgcacat ttggaaacct agcggcgacc ctgcctgctc
     5101 tcacagcata gcagttgccg acgaacactc ctctccggct caggcgttcg atctcgtacc
     5161 acactttgca gaccaggctc actgtgtttc tatctctctg tgcaggcagg aagctaaaaa
     5221 tgtgttcgac gacctcttca ggaaagtatg ccatggtggc ctcagaggcc agcttggggt
     5281 agttttcacg acaccttaaa tggaagaaaa aaactttgaa ccactgtctg aggcttgaga
     5341 atgaaccaag atccaaactc aaaaagggca aattccaagg agaattacat caagtgccaa
     5401 gctggcctaa cttcagtctc cacccactca gtatggggaa actccatcgc ataaaacccc
     5461 tccccccaac ctaaagacga cgtactccaa aagctcgaga actaatcgag gtgcctggac
     5521 ggcgcccggt actccgtgga gtcacatgaa gcgacggctg aggacggaaa ggcccttttc
     5581 ctttgtgtgg gtgactcacc cgcccgctct cccgagcgcc gcgtcctcca ttttgagctc
     5641 cctgcagcag ggccgggaag cggccatctt tccgctcacg caactggtgc cgaccgggcc
     5701 agccttgccg cccagggcgg ggcgatacac ggcggcgcga ggccaggcac cagagcaggc
     5761 cggccagctt gagactaccc ccgtccgatt ctcggtggcc gcgctcgcag gccccgcctc
     5821 gccgaacatg tgcgctggga cgcacgggcc ccgtcgccgc ccgcggcccc aaaaaccgaa
     5881 ataccagtgt gcagatcttg gcccgcattt acaagactat cttgccagaa aaaaagcgtc
     5941 gcagcaggtc atcaaaaatt ttaaatggct agagacttat cgaaagcagc gagacaggcg
     6001 cgaaggtgcc accagattcg cacgcggcgg ccccagcgcc caagccaggc ctcaactcaa
     6061 gcacgaggcg aaggggctcc ttaagcgcaa ggcctcgaac tctcccaccc acttccaacc
     6121 cgaagctcgg gatcaagaat cacgtactgc agccaggggc gtggaagtaa ttcaaggcac
     6181 gcaagggcca taacccgtaa agaggccagg cccgcgggaa ccacacacgg cacttacctg
     6241 tgttctggcg gcaaacccgt tgcgaaaaag aacgttcacg gcgactactg cacttatata
     6301 cggttctccc ccaccctcgg gaaaaaggcg gagccagtac acgacatcac tttcccagtt
     6361 taccccgcgc caccttctct aggcaccggt tcaattgccg acccctcccc ccaacttctc
     6421 ggggactgtg ggcgatgtgc gctctgccca ctgacgggca ccggagcctc taggcgagac
     6481 cctgtctcac aaaataaagt aagcccggac tgagtgcgga aaggcgggcc tggcgggtct
     6541 ggtctcccca tgcgggccac cagaggccct gcagccttca gtcgcttgaa ggggtaatgg
     6601 cgcttccact cacaaacatg gcggacagag cgtgtgaacg agatgaacag cccctcaaaa
     6661 atatggccgc cgaggctgga cggccgtgcc ccagcagcac cgcctccgcg ccccacgtga
     6721 tctctcgccg ggcacagcgc tgaccgcgga ggtccaaccg gaagaatgtc cggattggac
     6781 attcggaaga gggcccgcct tccctgggga atctctgcgc acgcgcagaa cgcttcgacc
     6841 aatgaaaaca caggaagccg tccgcgcaac cgcgttgcgt cacttctgcc gcccctgttt
     6901 caaggtatat agccgtagac ggaacttcgc ctttctctcg gccttagcgc catttttttg
     6961 ggtgagtgtt ttttggttcc tgcgttggga ttccgtgtac aatccataga catctgacct
     7021 cggcacttag catcatcaca gcaaactaac tgtagccttt ctctctttcc ctgtagaaac
     7081 ctctgcactc gacatatgtc tagattagat aaaagtaaag tgattaacag cgcattagag
     7141 ctgcttaatg aggtcggaat cgaaggttta acaacccgta aactcgccca gaagctaggt
     7201 gtagagcagc ctacattgta ttggcatgta aaaaataagc gggctttgct cgacgcctta
     7261 gccattgaga tgttagatag gcaccatact cacttttgcc ctttagaagg ggaaagctgg
     7321 caagattttt tacgtaataa cgctaaaagt tttagatgtg ctttactaag tcatcgcgat
     7381 ggagcaaaag tacatttagg tacacggcct acagaaaaac agtatgaaac tctcgaaaat
     7441 caattagcct ttttatgcca acaaggtttt tcactagaga atgcattata tgcactcagc
     7501 gctgtggggc attttacttt aggttgcgta ttggaagatc aagagcatca agtcgctaaa
     7561 gaagaaaggg aaacacctac tactgatagt atgccgccat tattacgaca agctatcgaa
     7621 ttatttgatc accaaggtgc agagccagcc ttcttattcg gccttgaatt gatcatatgt
     7681 ggcctggaga aacagctaaa gtgcgaaagc ggcgggccga ccgacgccct tgacgatttt
     7741 gacttagaca tgctcccagc cgatgccctt gacgactttg accttgatat gctgcctgct
     7801 gacgctcttg acgattttga ccttgacatg ctccccggat ctggagaggg cagaggaagt
     7861 ctgctaacat gcggtgacgt cgaggagaat cctggcccat cgatgattga acaagatgga
     7921 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     7981 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     8041 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg
     8101 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     8161 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     8221 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     8281 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     8341 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     8401 ccagccgaac tgttcgccag gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg
     8461 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgct
//
