LOCUS       sequence-376742-        8473 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 8473)
  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 8473)
  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: DHX35
FEATURES             Location/Qualifiers
     source          1..8473
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        98..1276
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          329..1267
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1224..1244
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     1293..1312
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1293..1311
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1390..1414
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(3527..3551)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3676..3717
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(4172..4189)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4178..4402
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4535..4554)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4686..4733
                     /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             4741..5763
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4741..5763
                     /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    5893..6014
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5930..5949)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5984..6003
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6063..6079)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6063..6079)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6076..6098)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6087..6103
                     /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(6111..6117,6118..6135,6136..6141))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6156..6177
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6294..6311)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6465..7053)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6545..6564)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7224..8015,8016..8084))
                     /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     7847..7866
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8085..8189)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8264..8283)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gcttttgcaa aaagctttgc aaagatggat aaagttttaa acagagagga atctttgcag
       61 ctaatggacc ttctaggtct tgaaaggagt gggaattggc tccggtgccc gtcagtgggc
      121 agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaaccgg
      181 tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct
      241 ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt
      301 tcgcaacggg tttgccgcca gaacacaggt aagtgccgtg tgtggttccc gcgggcctgg
      361 cctctttacg ggttatggcc cttgcgtgcc ttgaattact tccacctggc tgcagtacgt
      421 gattcttgat cccgagcttc gggttggaag tgggtgggag agttcgaggc cttgcgctta
      481 aggagcccct tcgcctcgtg cttgagttga ggcctggcct gggcgctggg gccgccgcgt
      541 gcgaatctgg tggcaccttc gcgcctgtct cgctgctttc gataagtctc tagccattta
      601 aaatttttga tgacctgctg cgacgctttt tttctggcaa gatagtcttg taaatgcggg
      661 ccaagatctg cacactggta tttcggtttt tggggccgcg ggcggcgacg gggcccgtgc
      721 gtcccagcgc acatgttcgg cgaggcgggg cctgcgagcg cggccaccga gaatcggacg
      781 ggggtagtct caagctggcc ggcctgctct ggtgcctggc ctcgcgccgc cgtgtatcgc
      841 cccgccctgg gcggcaaggc tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc
      901 gcttcccggc cctgctgcag ggagctcaaa atggaggacg cggcgctcgg gagagcgggc
      961 gggtgagtca cccacacaaa ggaaaagggc ctttccgtcc tcagccgtcg cttcatgtga
     1021 ctccacggag taccgggcgc cgtccaggca cctcgattag ttctcgagct tttggagtac
     1081 gtcgtcttta ggttgggggg aggggtttta tgcgatggag tttccccaca ctgagtgggt
     1141 ggagactgaa gttaggccag cttggcactt gatgtaattc tccttggaat ttgccctttt
     1201 tgagtttgga tcttggttca ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc
     1261 atttcaggtg tcgtgaggaa ttagcttggt actaatacga ctcactatag ggagacccaa
     1321 gctggctagg taagcttggt accgagctcg gatccactag tccagtgtgg tggaattctg
     1381 cagatatcaa caagtttgta caaaaaagtt ggcaccatgg ctgcgcccgt gggaccggtg
     1441 aagttctggc gacccggtac agaggggcca ggtgtaagca tctctgaaga gagacaaagt
     1501 ctggctgaaa actctgggac aacggttgtt tacaaccctt atgctgccct ttccatagag
     1561 cagcagaggc agaagctgcc ggtattcaag cttaggaatc atattttata cttgatagaa
     1621 aattatcaga cagtggtgat tgttggtgaa acaggatgtg ggaagagcac acagattcct
     1681 cagtaccttg cagaagccgg ctggacagct gaaggaagag tggtaggagt gacccagcct
     1741 cgaagagtgg ctgctgttac agttgcaggg agagtagctg aagaaagggg tgcagtgctg
     1801 ggccacgagg tgggctactg catccgcttt gatgactgca ccgaccagct ggccacgaga
     1861 attaagtttc ttactgatgg aatgctggtc agggaaatga tggttgatcc gttgttaaca
     1921 aaatatagtg tcatcatgct ggatgaagcc cacgagagga ccttgtacac tgacattgcc
     1981 attggcttgc taaaaaagat tcagaaaaag cgaggggatc ttcgattgat tgtagcttca
     2041 gccactctgg atgcagacaa attccgggat ttctttaatc aaaatgaaac cagtgatcca
     2101 gcaagggata catgtgtgat ccttacagtg gaagggagaa catttccggt ggatatcttt
     2161 tatctacaaa gtcctgttcc agattatatc aaatcaactg tcgaaactgt ggtgaaaatt
     2221 caccagacag agggagacgg agacgtttta gcatttctta ctggccagga agaggtagaa
     2281 actgttgtgt cgatgctcat cgagcaggct cgagcactag ctcgcactgg gatgaagaga
     2341 cacctccgag ttctccccat gtatgcagga ctgccttcct ttgagcaaat gaaagtgttt
     2401 gaaagggtgt cacgcagtgt cagaaaggtg atagtggcca ccaatgtggc agaaacctct
     2461 atcacaatca gcggcattgt gtatgtgatc gactgtggct ttgtgaaact ccgagcctac
     2521 aatcccagga cagctattga atgcttggtg gtggtgccag tctcccaggc atcagctaat
     2581 cagcgagcag gacgtggtgg tcgtagtcgc tcgggaaaat gttatcgcct ttatacagag
     2641 gaagcctttg acaagttgcc tcagtctacg gttcctgaga tgcagcgtag taatttggca
     2701 cctgtcatcc tgcagctgaa agcactagga attgacaatg tcctcaggtt ccacttcatg
     2761 tcgccccctc cagcacagtc gatggttcaa gccttggagt tactgtatgc tctgggaggt
     2821 ctggacaaag actgtcgcct aactgaaccg cttggcatga gaattgcaga gtttcctttg
     2881 aatcccatgt ttgccaaaat gctgcttgaa tcaggaaact tcggctgttc tcaggaaatt
     2941 ctaagcatcg ctgccatgat gcagatccag aatatctttg tggtcccccc aaaccagaag
     3001 tctcacgcaa ttcgagtgca ccgtaaattt gctgtggagg agggcgacca cctcactatg
     3061 ctcaatatat atgaagcatt tatcaaacac aataaggact ctaaatggtg tcaggaacat
     3121 ttcctgaatt acaagggtct tgtcagagct gcgactgtaa gagaacaatt gaaaaagctt
     3181 cttgtcaagt ttcaagtgcc caggaagtct agtgaaggtg acccggatct ggttctgagg
     3241 tgcattgtct ccggcttctt cgccaatgca gcgaggtttc attctactgg agcttatagg
     3301 accatccgtg atgaccatga gctgcacata caccctgcgt cagtcctcta tgcagagaag
     3361 ccgcctcgct gggtcatcta taacgaagtt atacagacct ccaagtacta catgagagat
     3421 gtgactgcca ttgaatcggc ctggctgttg gagctggctc cacactttta tcaacaagga
     3481 acgcacctgt ctctgaaagc caaaagggcc aaggtccagg acccgttgcc aactttcttg
     3541 tacaaagtgg ttgatatcca gcacagtggc ggccgctcga gtctagaggg cccgcggttc
     3601 gaaaccagcg gaggcggcgg aagcggggga ggaggcagcg gcggaggcgg cagcgggggc
     3661 ggcggaagcg ctaccggtaa gcctatccct aaccctctcc tcggtctcga ttctacgcgt
     3721 accggtatgg cttctaactt tactcagttc gttctcgtcg acaatggcgg aactggcgac
     3781 gtgactgtcg ccccaagcaa cttcgctaac ggggtcgctg aatggatcag ctctaactcg
     3841 cgatcacagg cttacaaagt aacctgtagc gttcgtcaga gctctgcgca gaatcgcaaa
     3901 tacaccatca aagtcgaggt gcctaaagtg gcaacccaga ctgttggtgg tgaagagctt
     3961 cctgtagccg gatggagatc ttacttaaat atggaactaa ccattccaat tttcgccacg
     4021 aattccgact gcgagcttat tgttaaggca atgcaaggtc tcctaaaaga tggaaacccg
     4081 attccctcgg ccatcgcggc caactccggc atctacggag gtggaggtgg atctcgtacg
     4141 ggttagtaat gagtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca
     4201 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     4261 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     4321 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     4381 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg
     4441 tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     4501 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     4561 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     4621 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     4681 acctagaagt tcctattccg aagttcctat tctctagaaa gtataggaac ttccttggcc
     4741 aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc
     4801 gtctccgacc tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta
     4861 ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt
     4921 tatgtttatc ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg
     4981 gaattcagcg agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa
     5041 gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg
     5101 atcgctgcgg ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc
     5161 ggtcaataca ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac
     5221 tggcaaactg tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg
     5281 atgctttggg ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc
     5341 aacaatgtcc tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg
     5401 ttcggggatt cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt
     5461 atggagcagc agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg
     5521 ctccgggcgt atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc
     5581 aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc
     5641 gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt
     5701 gtagaagtac tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa
     5761 tagcacgtac tacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     5821 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     5881 ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     5941 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     6001 atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca
     6061 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     6121 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     6181 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     6241 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     6301 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     6361 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     6421 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     6481 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     6541 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     6601 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     6661 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     6721 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     6781 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     6841 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     6901 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     6961 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     7021 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     7081 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     7141 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     7201 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     7261 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     7321 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     7381 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     7441 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     7501 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     7561 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     7621 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     7681 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     7741 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     7801 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     7861 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     7921 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     7981 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     8041 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     8101 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     8161 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     8221 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
     8281 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
     8341 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
     8401 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
     8461 gattattgac tag
//
