LOCUS       sequence-338224-        8327 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  constitutive expression of TIR1-myc and tTA in sleeping beauty 
            transposon vector with selection marker (puromycin).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8327)
  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 8327)
  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/Pur
FEATURES             Location/Qualifiers
     source          1..8327
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    102..326
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     315..332
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(389..418)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             complement(425..454)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             complement(461..490)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             complement(518..2242)
                     /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      2239..2248
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(2274..3456)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     primer_bind     complement(2306..2326)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     complement(4157..4176)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             4711..4821
                     /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             4834..4887
                     /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             4891..5490
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4891..4910)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5387..5407
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5590..5609)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5655..5710
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5709..5728)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(6102..6119)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6273..6861)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6353..6372)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7032..7823,7824..7892))
                     /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     7655..7674
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7893..7997)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 agtttacata cactaagttc gactcctctg cagaatgcgg cgatgtttcg gtaaggggtc
       61 cgctatctag taggcccagc tggttctttc cgcctcagaa gccatagagc ccaccgcatc
      121 cccagcatgc ctgctattgt cttcccaatc ctcccccttg ctgtcctgcc ccaccccacc
      181 ccccagaata gaatgacacc tactcagaca atgcgatgca atttcctcat tttattagga
      241 aaggacagtg ggagtggcac cttccagggt caaggaaggc acgggggagg ggcaaacaac
      301 agatggctgg caactagaag gcacagtcga ggctgatcag cgagctctag agaattgatc
      361 cccaagcttt ttagctagtg gatccgttca agtcttcttc tgagattaat ttttgttcac
      421 cgttcaagtc ttcctcggag attagctttt gttcaccgtt caaatcttct tcagaaatca
      481 acttttgttc accactagca gcagaaccgg agtcgactag gattttaaca aaatttggtg
      541 catcatccct cgccccagca gttgtgcggt acacatataa tttctccacc tttggcaggt
      601 cgccatgatt ttcctccatt tcgttagacc cgtcccgctc attaatgact tccacgttca
      661 gcataggcat tttactagcc aggacctgac agcccttcag agtcacattg caactagaca
      721 tccacaggct cctcattgtc tcgtatctgg caaagtttcc cagcagagcg gcatccccga
      781 aagggctgtc ccggatttcc agttttcgca gattcttaca cccgttcatc acgtgcatca
      841 tgcccttatc ggagtctccg gcaaatgcaa tgctcagcat ctccagctgc ttagcatact
      901 tgccgatgta catgaacact ttgtctgtca gcaggccgga aatgctcagc ctctggagtc
      961 ccttgcactc tctcacgata gctccaaacc cttcatccag tggctgggat gtgaccacgt
     1021 cgggtttgcc tggttccaga atacacagcc gaaatcgggt gaaattaggg cagttcttgg
     1081 cgacggtcac cagtgcagca ttagtcatct gatgacagaa atacagcagg ctgttcagct
     1141 ttgggcatcc cagtgagact gccaccaggc cttcctcagt gacagcacta tatccggcca
     1201 cgtaaaaatc agatgggaac actctcagct cctggaggtc cttgcaggag ctggcgacca
     1261 cctggagccc cttatcggaa atacagtcca gcacccacag tcgctggagt ttcacgcagc
     1321 gtgagatcat ctttgtcaga tcactggcgt ccagggttgg tgcatagctc aggttcaggc
     1381 ctgtcagctg tgcacacaga gggtagataa agctcaggca cacgggtgaa gcatcccaga
     1441 atcctgacag actcctcagc attttacact tctccagggc agaagtcagt ttaaagtagg
     1501 attctgtctg gaaatcgtct gtcagattgc cggttcccag atcctccagg ttaggggttc
     1561 tcagcagaat cttagccaga gtgtccacgc tcacgctccg gttcagccgc agagacctca
     1621 ggttggggct tcttgagacc agccgctcca gggacccagc attcacttcg cctttgatgc
     1681 aggcaaagtt cagggacacc aggctggtac atgagtctgg gaagcaagac agccatctgg
     1741 ggcctctatc ctccacttca ttctcctgga ggtccagctc ccgcagcagc ttacagtggg
     1801 aggccacggc tgccagtcca tcggtgctga agccttcgca agaaatcagc accagtgcgc
     1861 gaaacctagg gaagctccga gccagcagct ccagactttc gtcagagacc accattcgct
     1921 tcatgcgcag ttcctccagt ccatgacagc cgcgagcggc tgcctcgatc caagggccgg
     1981 cgtatcctcc ccaatcaggg ggcaccaggt taaagtcggc gaagtggggt ttccccttga
     2041 cggtcagtgc gcgcacattt ggaaacctag cggcgaccct gcctgctctc acagcatagc
     2101 agttgccgac gaacactcct ctccggctca ggcgttcgat ctcgtaccac actttgcaga
     2161 ccaggctcac tgtgtttcta tctctctgtg caggcaggaa gctaaaaatg tgttcgacga
     2221 cctcttcagg aaagtatgcc atggtggcct cagaggccag cttggggtag ttttcacgac
     2281 accttaaatg gaagaaaaaa actttgaacc actgtctgag gcttgagaat gaaccaagat
     2341 ccaaactcaa aaagggcaaa ttccaaggag aattacatca agtgccaagc tggcctaact
     2401 tcagtctcca cccactcagt atggggaaac tccatcgcat aaaacccctc cccccaacct
     2461 aaagacgacg tactccaaaa gctcgagaac taatcgaggt gcctggacgg cgcccggtac
     2521 tccgtggagt cacatgaagc gacggctgag gacggaaagg cccttttcct ttgtgtgggt
     2581 gactcacccg cccgctctcc cgagcgccgc gtcctccatt ttgagctccc tgcagcaggg
     2641 ccgggaagcg gccatctttc cgctcacgca actggtgccg accgggccag ccttgccgcc
     2701 cagggcgggg cgatacacgg cggcgcgagg ccaggcacca gagcaggccg gccagcttga
     2761 gactaccccc gtccgattct cggtggccgc gctcgcaggc cccgcctcgc cgaacatgtg
     2821 cgctgggacg cacgggcccc gtcgccgccc gcggccccaa aaaccgaaat accagtgtgc
     2881 agatcttggc ccgcatttac aagactatct tgccagaaaa aaagcgtcgc agcaggtcat
     2941 caaaaatttt aaatggctag agacttatcg aaagcagcga gacaggcgcg aaggtgccac
     3001 cagattcgca cgcggcggcc ccagcgccca agccaggcct caactcaagc acgaggcgaa
     3061 ggggctcctt aagcgcaagg cctcgaactc tcccacccac ttccaacccg aagctcggga
     3121 tcaagaatca cgtactgcag ccaggggcgt ggaagtaatt caaggcacgc aagggccata
     3181 acccgtaaag aggccaggcc cgcgggaacc acacacggca cttacctgtg ttctggcggc
     3241 aaacccgttg cgaaaaagaa cgttcacggc gactactgca cttatatacg gttctccccc
     3301 accctcggga aaaaggcgga gccagtacac gacatcactt tcccagttta ccccgcgcca
     3361 ccttctctag gcaccggttc aattgccgac ccctcccccc aacttctcgg ggactgtggg
     3421 cgatgtgcgc tctgcccact gacgggcacc ggagcctcta ggcgagaccc tgtctcacaa
     3481 aataaagtaa gcccggactg agtgcggaaa ggcgggcctg gcgggtctgg tctccccatg
     3541 cgggccacca gaggccctgc agccttcagt cgcttgaagg ggtaatggcg cttccactca
     3601 caaacatggc ggacagagcg tgtgaacgag atgaacagcc cctcaaaaat atggccgccg
     3661 aggctggacg gccgtgcccc agcagcaccg cctccgcgcc ccacgtgatc tctcgccggg
     3721 cacagcgctg accgcggagg tccaaccgga agaatgtccg gattggacat tcggaagagg
     3781 gcccgccttc cctggggaat ctctgcgcac gcgcagaacg cttcgaccaa tgaaaacaca
     3841 ggaagccgtc cgcgcaaccg cgttgcgtca cttctgccgc ccctgtttca aggtatatag
     3901 ccgtagacgg aacttcgcct ttctctcggc cttagcgcca tttttttggg tgagtgtttt
     3961 ttggttcctg cgttgggatt ccgtgtacaa tccatagaca tctgacctcg gcacttagca
     4021 tcatcacagc aaactaactg tagcctttct ctctttccct gtagaaacct ctgcactcga
     4081 catatgtcta gattagataa aagtaaagtg attaacagcg cattagagct gcttaatgag
     4141 gtcggaatcg aaggtttaac aacccgtaaa ctcgcccaga agctaggtgt agagcagcct
     4201 acattgtatt ggcatgtaaa aaataagcgg gctttgctcg acgccttagc cattgagatg
     4261 ttagataggc accatactca cttttgccct ttagaagggg aaagctggca agatttttta
     4321 cgtaataacg ctaaaagttt tagatgtgct ttactaagtc atcgcgatgg agcaaaagta
     4381 catttaggta cacggcctac agaaaaacag tatgaaactc tcgaaaatca attagccttt
     4441 ttatgccaac aaggtttttc actagagaat gcattatatg cactcagcgc tgtggggcat
     4501 tttactttag gttgcgtatt ggaagatcaa gagcatcaag tcgctaaaga agaaagggaa
     4561 acacctacta ctgatagtat gccgccatta ttacgacaag ctatcgaatt atttgatcac
     4621 caaggtgcag agccagcctt cttattcggc cttgaattga tcatatgtgg cctggagaaa
     4681 cagctaaagt gcgaaagcgg cgggccgacc gacgcccttg acgattttga cttagacatg
     4741 ctcccagccg atgcccttga cgactttgac cttgatatgc tgcctgctga cgctcttgac
     4801 gattttgacc ttgacatgct ccccggatct ggagagggca gaggaagtct gctaacatgc
     4861 ggtgacgtcg aggagaatcc tggcccatcg atgaccgagt acaagcccac ggtgcgcctc
     4921 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac
     4981 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa
     5041 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
     5101 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     5161 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa
     5221 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc
     5281 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg
     5341 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc
     5401 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc
     5461 tggtgcatga cccgcaagcc cggtgcctga ttcgaaggcc tgtcgtgaat tcactcctca
     5521 ggtgcaggct gcctatcaga aggtggtggc tggtgtggcc aatgccctgg ctcacaaata
     5581 ccactgagat ctttttccct ctgccaaaaa ttatggggac atcatgaagc cccttgagca
     5641 tctgacttct ggctaataaa ggaaatttat tttcattgca atagtgtgtt ggaatttttt
     5701 gtgtctctca ctcggaagga catatgggag ggcaaatcat ttaaaacatc agaatgagta
     5761 tttggtttag agtttggcaa catatgccat atgctggctg ccatgaacta gctactcggg
     5821 accccttacc gaaacatcgc cgcattctgc agaggagtcg agtgtatgta aacttctgac
     5881 ccactgggaa tgtgatgaaa gaaataaaag ctgaaatgaa tcattctctc tactattatt
     5941 ctgatatttc acattcttaa aataaagtgg tgatcctaac tgacctaaga cagggaattt
     6001 ttactaggat taaatgtcag gaattgtgaa aaagtgagtt taaatgtatt tggctaaggt
     6061 gtatgtaaac ttccgacttc aactgtatag ggatccgctt cctcgctcac tgactcgctg
     6121 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     6181 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     6241 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     6301 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     6361 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     6421 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     6481 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     6541 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     6601 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     6661 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     6721 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     6781 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     6841 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     6901 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     6961 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     7021 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     7081 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     7141 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     7201 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     7261 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     7321 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     7381 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     7441 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     7501 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     7561 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     7621 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     7681 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     7741 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     7801 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     7861 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     7921 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     7981 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgatgcggt gtgaaatacc
     8041 gcacagatgc gtaaggagaa aataccgcat caggaaattg taagcgttaa tattttgtta
     8101 aaattggatc cctatacagt tgaagtcgga agtttacata cacttaagtt ggagtcatta
     8161 aaactcgttt ttcaactact ccacaaattt cttgttaaca aacaatagtt ttggcaagtc
     8221 agttaggaca tctactttgt gcatgacaca agtcattttt ccaacaattg tttacagaca
     8281 gattatttca cttataattc actgtatcac aattccagtg ggtcaga
//
