LOCUS       sequence-377219-        8410 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 8410)
  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 8410)
  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: THOC5
FEATURES             Location/Qualifiers
     source          1..8410
                     /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"
     CDS             complement(2115..2126)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    complement(3367..3391)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3516..3557
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(4012..4029)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4018..4242
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4375..4394)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4526..4573
                     /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             4581..5603
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4581..5603
                     /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    5733..5854
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5770..5789)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5824..5843
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5903..5919)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5903..5919)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5916..5938)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5927..5943
                     /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(5951..5957,5958..5975,5976..5981))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5996..6017
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6134..6151)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6305..6893)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6385..6404)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7064..7855,7856..7924))
                     /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     7687..7706
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7925..8029)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8104..8123)
                     /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 gttggcacca
     1321 tgtcatcaga atcgagcaaa aaacggaagc ccaaagtgat ccgaagcgat ggagccccag
     1381 ctgaaggaaa gcggaatcga tctgacaccg agcaggaagg taaatactac agtgaggagg
     1441 ccgaggtgga tctgcgggac cctggcagag actatgagtt atacaagtac acctgccagg
     1501 agctacagag gctgatggct gagatccaag acctgaagag caggggtggc aaggatgtgg
     1561 caatagaaat agaagaacgg aggatccaga gctgtgtgca tttcatgact ctaaagaagc
     1621 ttaaccgatt agcccacatc aggttgaaga aaggaagaga tcagacccac gaggctaagc
     1681 agaaagtaga tgcctatcat ctgcagctcc agaacctgtt gtatgaggtg atgcacctac
     1741 agaaggagat caccaaatgt ttggagttta agtcaaagca tgaagaaatt gatctggtca
     1801 gtttagagga gttttataag gaggctccac cagatatcag caaggccgaa gtcaccatgg
     1861 gagaccctca ccagcaaaca ctggcacgtc tggactggga gctggagcag cggaaaaggc
     1921 tggcagagaa gtaccgagag tgcctatcta acaaggagaa gattctcaag gagattgagg
     1981 tgaagaagga gtacctgagc agcctccagc cccgcctcaa cagcatcatg caggcttccc
     2041 ttccggtgca ggagtacctg tttatgccat tcgaccaggc tcacaagcag tatgagacag
     2101 ccagacacct gccgcctccc ctctatgtcc tctttgttca ggccactgcg tatgggcagg
     2161 cctgtgataa gacgttatct gtggcaatcg aaggcagtgt ggatgaagcc aaggctctgt
     2221 tcaaacctcc agaggactcc caagatgacg agagtgactc agatgccgag gaggagcaga
     2281 ctacgaagcg ccggagaccc acactggggg ttcagttgga cgacaaacgc aaggagatgc
     2341 tgaagaggca cccactgtct gtcatgctcg acctgaagtg caaagatgac agtgtgcttc
     2401 acctgacttt ctactacctc atgaacctca acatcatgac agtaaaagcc aaagtgacaa
     2461 ctgccatgga gctgatcacc cccatcagtg caggtgactt gctgtctcct gactcagtcc
     2521 tgagttgctt gtatcctggg gatcatggaa agaaaactcc gaatccagcc aatcagtatc
     2581 agtttgataa agttggcatc ctgactttga gcgactatgt acttgagcta ggtcacccct
     2641 atttgtgggt gcagaagctg ggtggcctcc acttccccaa agagcagccc cagcaaacag
     2701 tgattgctga ccactcgctg agcgccagcc acatggagac caccatgaaa cttctgaaga
     2761 ccagggtgca gtcccgcctg gccctccaca aacagtttgc atccctagaa catggcattg
     2821 tgccagttac cagtgattgc cagtacctct tccctgccaa ggttgtctct cgcctggtga
     2881 aatgggtgac aattgcccat gaggattaca tggagctgca cttcaccaaa gacattgtgg
     2941 atgcgggact ggctggggac accaatctct actacatggc gctcatcgaa aggggcacag
     3001 ccaaactgca ggccgctgtg gtgttgaacc ctggctactc ctccatccca cctattttcc
     3061 agctctgttt gaactggaaa ggggagaaaa ccaacagcaa cgatgacaac attcgggcca
     3121 tggagggcga agtcaatgtg tgctacaagg agctgtgtgg cccttggccc agccaccagc
     3181 tgttgaccaa ccagctgcag cggctgtgtg tgctgctgga tgtttacctg gagaccgaga
     3241 gccatgacga cagtgtggag gggcccaagg aatttcccca ggagaagatg tgtctgcggc
     3301 tcttcagggg tcctagcagg atgaagccat ttaaatacaa ccatcctcag ggattcttca
     3361 gccatcgccc aactttcttg tacaaagtgg ttgatatcca gcacagtggc ggccgctcga
     3421 gtctagaggg cccgcggttc gaaaccagcg gaggcggcgg aagcggggga ggaggcagcg
     3481 gcggaggcgg cagcgggggc ggcggaagcg ctaccggtaa gcctatccct aaccctctcc
     3541 tcggtctcga ttctacgcgt accggtatgg cttctaactt tactcagttc gttctcgtcg
     3601 acaatggcgg aactggcgac gtgactgtcg ccccaagcaa cttcgctaac ggggtcgctg
     3661 aatggatcag ctctaactcg cgatcacagg cttacaaagt aacctgtagc gttcgtcaga
     3721 gctctgcgca gaatcgcaaa tacaccatca aagtcgaggt gcctaaagtg gcaacccaga
     3781 ctgttggtgg tgaagagctt cctgtagccg gatggagatc ttacttaaat atggaactaa
     3841 ccattccaat tttcgccacg aattccgact gcgagcttat tgttaaggca atgcaaggtc
     3901 tcctaaaaga tggaaacccg attccctcgg ccatcgcggc caactccggc atctacggag
     3961 gtggaggtgg atctcgtacg ggttagtaat gagtttaaac ccgctgatca gcctcgactg
     4021 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     4081 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     4141 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     4201 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     4261 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
     4321 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     4381 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     4441 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     4501 attagggtga tggttcacgt acctagaagt tcctattccg aagttcctat tctctagaaa
     4561 gtataggaac ttccttggcc aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc
     4621 tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc gaagaatctc
     4681 gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg
     4741 atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg ctcccgattc
     4801 cggaagtgct tgacattggg gaattcagcg agagcctgac ctattgcatc tcccgccgtg
     4861 cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg
     4921 tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc gggttcggcc
     4981 cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata tgcgcgattg
     5041 ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt gcgtccgtcg
     5101 cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc cggcacctcg
     5161 tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata acagcggtca
     5221 ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac atcttcttct
     5281 ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg aggcatccgg
     5341 agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt gaccaactct
     5401 atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg
     5461 caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg
     5521 ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga cgccccagca
     5581 ctcgtccgag ggcaaaggaa tagcacgtac tacgagattt cgattccacc gccgccttct
     5641 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     5701 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     5761 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     5821 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     5881 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     5941 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     6001 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     6061 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     6121 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     6181 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     6241 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     6301 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     6361 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     6421 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     6481 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     6541 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     6601 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     6661 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     6721 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     6781 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     6841 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     6901 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     6961 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     7021 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     7081 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     7141 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     7201 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     7261 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     7321 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     7381 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     7441 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     7501 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     7561 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     7621 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     7681 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     7741 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     7801 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     7861 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     7921 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     7981 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     8041 gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct
     8101 cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
     8161 ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga
     8221 caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
     8281 cagatatacg cgttgacatt gattattgac taggcttttg caaaaagctt tgcaaagatg
     8341 gataaagttt taaacagaga ggaatctttg cagctaatgg accttctagg tcttgaaagg
     8401 agtgggaatt
//
