LOCUS       sequence-377077-        7414 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 7414)
  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 7414)
  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: THOC3
FEATURES             Location/Qualifiers
     source          1..7414
                     /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(2371..2395)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2520..2561
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3016..3033)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3022..3246
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3379..3398)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3530..3577
                     /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             3585..4607
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3585..4607
                     /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    4737..4858
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4774..4793)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4828..4847
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4907..4923)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4907..4923)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4920..4942)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4931..4947
                     /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(4955..4961,4962..4979,4980..4985))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5000..5021
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5138..5155)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5309..5897)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5389..5408)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6068..6859,6860..6928))
                     /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     6691..6710
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6929..7033)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7108..7127)
                     /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 gttggcatgg
     1321 cggtccccgc tgcagccatg gggccctcgg cgttgggcca gagcggcccc ggctcgatgg
     1381 ccccgtggtg ctcagtgagc agcggcccgt cgcgctacgt gcttgggatg caggagctgt
     1441 tccggggcca cagcaagacg cgcgagttcc tggcgcacag cgccaaggtg cactcggtgg
     1501 cctggagttg cgacgggcgt cgcctagcct cggggtcctt cgacaagacg gccagcgtct
     1561 tcttgctgga gaaggaccgg ttggtcaaag aaaacaatta tcggggacat ggggatagtg
     1621 tggaccagct ttgttggcat ccaagtaatc ctgacctatt tgttacggcg tctggagata
     1681 aaaccattcg catctgggat gtgaggacta caaaatgcat tgccactgtg aacactaaag
     1741 gggagaacat taatatctgc tggagtcctg atgggcagac cattgctgta ggcaacaagg
     1801 atgatgtggt gacctttatt gatgccaaga cacaccgttc caaagcagaa gagcagttca
     1861 agttcgaggt caacgaaatc tcctggaaca atgacaataa tatgttcttc ctgacaaatg
     1921 gcaatggttg tatcaacatc ctcagctacc cagaactgaa gcctgtgcag tccatcaacg
     1981 cccatccttc caactgcatc tgtatcaagt ttgaccccat ggggaagtac tttgccacag
     2041 gaagtgcaga tgctttggtc agcctctggg atgtggatga gttagtgtgt gttcggtgct
     2101 tttccaggct ggattggcct gtaagaaccc tcagtttcag ccatgatggg aaaatgctgg
     2161 cgtcagcatc ggaagatcat tttattgaca ttgctgaagt ggagacaggg gacaaactat
     2221 gggaggtaca gtgtgagtct ccgaccttca cagtggcgtg gcaccccaaa aggcctctgc
     2281 tggcatttgc ctgtgatgac aaagacggca aatatgacag cagccgggaa gccggaactg
     2341 tgaagctgtt tgggcttcct aatgattctt gcccaacttt cttgtacaaa gtggttgata
     2401 tccagcacag tggcggccgc tcgagtctag agggcccgcg gttcgaaacc agcggaggcg
     2461 gcggaagcgg gggaggaggc agcggcggag gcggcagcgg gggcggcgga agcgctaccg
     2521 gtaagcctat ccctaaccct ctcctcggtc tcgattctac gcgtaccggt atggcttcta
     2581 actttactca gttcgttctc gtcgacaatg gcggaactgg cgacgtgact gtcgccccaa
     2641 gcaacttcgc taacggggtc gctgaatgga tcagctctaa ctcgcgatca caggcttaca
     2701 aagtaacctg tagcgttcgt cagagctctg cgcagaatcg caaatacacc atcaaagtcg
     2761 aggtgcctaa agtggcaacc cagactgttg gtggtgaaga gcttcctgta gccggatgga
     2821 gatcttactt aaatatggaa ctaaccattc caattttcgc cacgaattcc gactgcgagc
     2881 ttattgttaa ggcaatgcaa ggtctcctaa aagatggaaa cccgattccc tcggccatcg
     2941 cggccaactc cggcatctac ggaggtggag gtggatctcg tacgggttag taatgagttt
     3001 aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     3061 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     3121 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     3181 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     3241 ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct
     3301 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     3361 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     3421 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     3481 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtacctag aagttcctat
     3541 tccgaagttc ctattctcta gaaagtatag gaacttcctt ggccaaaaag cctgaactca
     3601 ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc gacctgatgc
     3661 agctctcgga gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg
     3721 tcctgcgggt aaatagctgc gccgatggtt tctacaaaga tcgttatgtt tatcggcact
     3781 ttgcatcggc cgcgctcccg attccggaag tgcttgacat tggggaattc agcgagagcc
     3841 tgacctattg catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg
     3901 aactgcccgc tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc
     3961 ttagccagac gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa tacactacat
     4021 ggcgtgattt catatgcgcg attgctgatc cccatgtgta tcactggcaa actgtgatgg
     4081 acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg
     4141 actgccccga agtccggcac ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg
     4201 acaatggccg cataacagcg gtcattgact ggagcgaggc gatgttcggg gattcccaat
     4261 acgaggtcgc caacatcttc ttctggaggc cgtggttggc ttgtatggag cagcagacgc
     4321 gctacttcga gcggaggcat ccggagcttg caggatcgcc gcggctccgg gcgtatatgc
     4381 tccgcattgg tcttgaccaa ctctatcaga gcttggttga cggcaatttc gatgatgcag
     4441 cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg agccgggact gtcgggcgta
     4501 cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg
     4561 atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa ggaatagcac gtactacgag
     4621 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     4681 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact
     4741 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     4801 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     4861 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     4921 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     4981 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     5041 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     5101 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     5161 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     5221 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     5281 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     5341 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     5401 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     5461 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     5521 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     5581 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     5641 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     5701 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     5761 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     5821 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     5881 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     5941 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     6001 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     6061 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     6121 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     6181 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     6241 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     6301 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     6361 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     6421 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     6481 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     6541 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     6601 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     6661 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     6721 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     6781 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     6841 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     6901 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     6961 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     7021 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc
     7081 tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     7141 gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc
     7201 tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt
     7261 tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat tgactaggct
     7321 tttgcaaaaa gctttgcaaa gatggataaa gttttaaaca gagaggaatc tttgcagcta
     7381 atggaccttc taggtcttga aaggagtggg aatt
//
