LOCUS       sequence-377223-        7396 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For use in RBP tethering screen.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7396)
  AUTHORS   Luo EC, Nathanson JL, Tan FE, Schwartz JL, Schmok JC, Shankar A, 
            Markmiller S, Yee BA, Sathe S, Pratt GA, Scaletta DB, Ha Y, Hill DE,
            Aigner S, Yeo GW
  TITLE     Large-scale tethered function assays identify factors that regulate 
            mRNA stability and translation.
  JOURNAL   Nat Struct Mol Biol. 2020 Oct;27(10):989-1000. doi: 
            10.1038/s41594-020-0477-6. Epub 2020 Aug 17.
  PUBMED    32807991
REFERENCE   2  (bases 1 to 7396)
  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"; doi: 
            "10.1038/s41594-020-0477-6"; journalName: "Nat Struct Mol Biol"; 
            date: "2020-10"; volume: "27"; issue: "10"; pages: "989-1000"
COMMENT     Sequence Label: TOB1
FEATURES             Location/Qualifiers
     source          1..7396
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..1179
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          232..1170
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1127..1147
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     1196..1215
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1196..1214
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1293..1317
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2353..2377)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2502..2543
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2998..3015)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3004..3228
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3361..3380)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3512..3559
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3567..4589
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3567..4589
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4719..4840
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4756..4775)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4810..4829
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4889..4905)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4889..4905)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4902..4924)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4913..4929
                     /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)."
     promoter        complement(join(4937..4943,4944..4961,4962..4967))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4982..5003
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5120..5137)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5291..5879)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5371..5390)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6050..6841,6842..6910))
                     /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     6673..6692
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6911..7015)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7090..7109)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg
       61 ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt
      121 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca
      181 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc
      241 gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt
      301 acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
      361 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
      421 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
      481 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
      541 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
      601 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
      661 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
      721 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
      781 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
      841 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
      901 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
      961 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     1021 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     1081 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     1141 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gaattagctt ggtactaata
     1201 cgactcacta tagggagacc caagctggct aggtaagctt ggtaccgagc tcggatccac
     1261 tagtccagtg tggtggaatt ctgcagatat caacaagttt gtacaaaaaa gttggcatgc
     1321 agcttgaaat ccaagtagca ctaaatttta ttatttcgta tttgtacaat aagcttccca
     1381 ggagacgtgt caacattttt ggtgaagaac ttgaaagact tcttaagaag aaatatgaag
     1441 ggcactggta tcctgaaaag ccatacaaag gatcggggtt tagatgtata cacatagggg
     1501 agaaagtgga cccagtgatt gaacaagcat ccaaagagag tggtttggac attgatgatg
     1561 ttcgtggcaa tctgccacag gatcttagtg tttggatcga cccatttgag gtttcttacc
     1621 aaattggtga aaagggacca gtgaaggtgc tttacgtgga tgataataat gaaaatggat
     1681 gtgagttgga taaggagatc aaaaacagct ttaacccaga ggcccaggtt tttatgccca
     1741 taagtgaccc agcctcatca gtgtccagct ctccatcgcc tccttttggt cactctgctg
     1801 ctgtaagccc taccttcatg ccccggtcca ctcagccttt aacctttacc actgccactt
     1861 ttgctgccac caagttcggc tctaccaaaa tgaagaatag tggccgtagc aacaaggttg
     1921 cacgtacttc tcccatcaac ctcggcttga atgtgaatga cctcttgaag cagaaagcca
     1981 tctcttcctc aatgcactct ctgtatgggc ttggcttggg tagccagcag cagccacagc
     2041 aacagcagca gccagcccag ccgccaccgc caccaccacc accacagcag caacaacagc
     2101 agaaaacctc tgctctttct cctaatgcca aggaatttat ttttcctaat atgcagggtc
     2161 aaggtagtag taccaatgga atgttcccag gtgacagccc ccttaacctc agtcctctcc
     2221 agtacagtaa tgcctttgat gtgtttgcag cctatggagg cctcaatgag aagtcttttg
     2281 tagatggctt gaattttagc ttaaataaca tgcagtattc taaccagcaa ttccagcctg
     2341 ttatggctaa ctacccaact ttcttgtaca aagtggttga tatccagcac agtggcggcc
     2401 gctcgagtct agagggcccg cggttcgaaa ccagcggagg cggcggaagc gggggaggag
     2461 gcagcggcgg aggcggcagc gggggcggcg gaagcgctac cggtaagcct atccctaacc
     2521 ctctcctcgg tctcgattct acgcgtaccg gtatggcttc taactttact cagttcgttc
     2581 tcgtcgacaa tggcggaact ggcgacgtga ctgtcgcccc aagcaacttc gctaacgggg
     2641 tcgctgaatg gatcagctct aactcgcgat cacaggctta caaagtaacc tgtagcgttc
     2701 gtcagagctc tgcgcagaat cgcaaataca ccatcaaagt cgaggtgcct aaagtggcaa
     2761 cccagactgt tggtggtgaa gagcttcctg tagccggatg gagatcttac ttaaatatgg
     2821 aactaaccat tccaattttc gccacgaatt ccgactgcga gcttattgtt aaggcaatgc
     2881 aaggtctcct aaaagatgga aacccgattc cctcggccat cgcggccaac tccggcatct
     2941 acggaggtgg aggtggatct cgtacgggtt agtaatgagt ttaaacccgc tgatcagcct
     3001 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     3061 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     3121 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     3181 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg
     3241 aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg
     3301 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     3361 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     3421 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     3481 aacttgatta gggtgatggt tcacgtacct agaagttcct attccgaagt tcctattctc
     3541 tagaaagtat aggaacttcc ttggccaaaa agcctgaact caccgcgacg tctgtcgaga
     3601 agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag
     3661 aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct
     3721 gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc
     3781 cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc
     3841 gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc
     3901 agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt
     3961 tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg
     4021 cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt
     4081 ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc
     4141 acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag
     4201 cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct
     4261 tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc
     4321 atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc
     4381 aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat
     4441 gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa
     4501 gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc
     4561 ccagcactcg tccgagggca aaggaatagc acgtactacg agatttcgat tccaccgccg
     4621 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     4681 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     4741 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     4801 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     4861 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     4921 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     4981 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5041 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5101 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     5161 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     5221 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5281 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     5341 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     5401 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     5461 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     5521 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     5581 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     5641 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     5701 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     5761 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     5821 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     5881 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     5941 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     6001 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6061 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6121 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6181 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     6241 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     6301 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     6361 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     6421 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     6481 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     6541 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     6601 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     6661 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     6721 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     6781 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     6841 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     6901 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     6961 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     7021 ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg
     7081 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg
     7141 tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt
     7201 gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt
     7261 acgggccaga tatacgcgtt gacattgatt attgactagg cttttgcaaa aagctttgca
     7321 aagatggata aagttttaaa cagagaggaa tctttgcagc taatggacct tctaggtctt
     7381 gaaaggagtg ggaatt
//
