LOCUS       sequence-376740-        8011 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 8011)
  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 8011)
  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: DDX56
FEATURES             Location/Qualifiers
     source          1..8011
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        125..1303
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          356..1294
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1251..1271
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     1320..1339
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1320..1338
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1417..1441
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(3092..3116)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3241..3282
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3737..3754)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3743..3967
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4100..4119)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4251..4298
                     /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             4306..5328
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4306..5328
                     /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    5458..5579
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5495..5514)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5549..5568
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5628..5644)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5628..5644)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5641..5663)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5652..5668
                     /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(5676..5682,5683..5700,5701..5706))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5721..5742
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5859..5876)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6030..6618)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6110..6129)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6789..7580,7581..7649))
                     /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     7412..7431
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7650..7754)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7829..7848)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tacgcgttga cattgattat tgactaggct tttgcaaaaa gctttgcaaa gatggataaa
       61 gttttaaaca gagaggaatc tttgcagcta atggaccttc taggtcttga aaggagtggg
      121 aattggctcc ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt
      181 ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg taaactggga
      241 aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag
      301 tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggtaag
      361 tgccgtgtgt ggttcccgcg ggcctggcct ctttacgggt tatggccctt gcgtgccttg
      421 aattacttcc acctggctgc agtacgtgat tcttgatccc gagcttcggg ttggaagtgg
      481 gtgggagagt tcgaggcctt gcgcttaagg agccccttcg cctcgtgctt gagttgaggc
      541 ctggcctggg cgctggggcc gccgcgtgcg aatctggtgg caccttcgcg cctgtctcgc
      601 tgctttcgat aagtctctag ccatttaaaa tttttgatga cctgctgcga cgcttttttt
      661 ctggcaagat agtcttgtaa atgcgggcca agatctgcac actggtattt cggtttttgg
      721 ggccgcgggc ggcgacgggg cccgtgcgtc ccagcgcaca tgttcggcga ggcggggcct
      781 gcgagcgcgg ccaccgagaa tcggacgggg gtagtctcaa gctggccggc ctgctctggt
      841 gcctggcctc gcgccgccgt gtatcgcccc gccctgggcg gcaaggctgg cccggtcggc
      901 accagttgcg tgagcggaaa gatggccgct tcccggccct gctgcaggga gctcaaaatg
      961 gaggacgcgg cgctcgggag agcgggcggg tgagtcaccc acacaaagga aaagggcctt
     1021 tccgtcctca gccgtcgctt catgtgactc cacggagtac cgggcgccgt ccaggcacct
     1081 cgattagttc tcgagctttt ggagtacgtc gtctttaggt tggggggagg ggttttatgc
     1141 gatggagttt ccccacactg agtgggtgga gactgaagtt aggccagctt ggcacttgat
     1201 gtaattctcc ttggaatttg ccctttttga gtttggatct tggttcattc tcaagcctca
     1261 gacagtggtt caaagttttt ttcttccatt tcaggtgtcg tgaggaatta gcttggtact
     1321 aatacgactc actataggga gacccaagct ggctaggtaa gcttggtacc gagctcggat
     1381 ccactagtcc agtgtggtgg aattctgcag atatcaacaa gtttgtacaa aaaagcaggc
     1441 tccaccatgg aggactctga agcactgggc ttcgaacaca tgggcctcga tccccggctc
     1501 cttcaggctg tcaccgatct gggctggtcg cgacctacgc tgatccagga gaaggccatc
     1561 ccactggccc tagaagggaa ggacctcctg gctcgggccc gcacgggctc cgggaagacg
     1621 gccgcttatg ctattccgat gctgcagctg ttgctccata ggaaggcgac aggtccggtg
     1681 gtagaacagg cagtgagagg ccttgttctt gttcctacca aggagctggc acggcaagca
     1741 cagtccatga ttcagcagct ggctacctac tgtgctcggg atgtccgagt ggccaatgtc
     1801 tcagctgctg aagactcagt ctctcagaga gctgtgctga tggagaagcc agatgtggta
     1861 gtagggaccc catctcgcat attaagccac ttgcagcaag acagcctgaa acttcgtgac
     1921 tccctggagc ttttggtggt ggacgaagct gaccttcttt tttcctttgg ctttgaagaa
     1981 gagctcaaga gtctcctctg tcacttgccc cggatttacc aggcttttct catgtcagct
     2041 acttttaacg aggacgtaca agcactcaag gagctgatat tacataaccc ggttaccctt
     2101 aagttacagg agtcccagct gcctgggcca gaccagttac agcagtttca ggtggtctgt
     2161 gagactgagg aagacaaatt cctcctgctg tatgccctgc tcaagctgtc attgattcgg
     2221 ggcaagtctc tgctctttgt caacactcta gaacggagtt accggctacg cctgttcttg
     2281 gaacagttca gcatccccac ctgtgtgctc aatggagagc ttccactgcg ctccaggtgc
     2341 cacatcatct cacagttcaa ccaaggcttc tacgactgtg tcatagcaac tgatgctgaa
     2401 gtcctggggg ccccagtcaa gggcaagcgt cggggccgag ggcccaaagg ggacaaggcc
     2461 tctgatccgg aagcaggtgt ggcccggggc atagacttcc accatgtgtc tgctgtgctc
     2521 aactttgatc ttcccccaac ccctgaggcc tacatccatc gagctggcag gacagcacgc
     2581 gctaacaacc caggcatagt cttaaccttt gtgcttccca cggagcagtt ccacttaggc
     2641 aagattgagg agcttctcag tggagagaac aggggcccca ttctgctccc ctaccagttc
     2701 cggatggagg agatcgaggg cttccgctat cgctgcaggg atgccatgcg ctcagtgact
     2761 aagcaggcca ttcgggaggc aagattgaag gagatcaagg aagagcttct gcattctgag
     2821 aagcttaaga catactttga agacaaccct agggacctcc agctgctgcg gcatgaccta
     2881 cctttgcacc ccgcagtggt gaagccccac ctgggccatg ttcctgacta cctggttcct
     2941 cctgctctcc gtggcctggt acgccctcac aagaagcgga agaagctgtc ttcctcttgt
     3001 aggaaggcca agagagcaaa gtcccagaac ccactgcgca gcttcaagca caaaggaaag
     3061 aaattcagac ccacagccaa gccctccttg gacccagctt tcttgtacaa agtggttgat
     3121 atccagcaca gtggcggccg ctcgagtcta gagggcccgc ggttcgaaac cagcggaggc
     3181 ggcggaagcg ggggaggagg cagcggcgga ggcggcagcg ggggcggcgg aagcgctacc
     3241 ggtaagccta tccctaaccc tctcctcggt ctcgattcta cgcgtaccgg tatggcttct
     3301 aactttactc agttcgttct cgtcgacaat ggcggaactg gcgacgtgac tgtcgcccca
     3361 agcaacttcg ctaacggggt cgctgaatgg atcagctcta actcgcgatc acaggcttac
     3421 aaagtaacct gtagcgttcg tcagagctct gcgcagaatc gcaaatacac catcaaagtc
     3481 gaggtgccta aagtggcaac ccagactgtt ggtggtgaag agcttcctgt agccggatgg
     3541 agatcttact taaatatgga actaaccatt ccaattttcg ccacgaattc cgactgcgag
     3601 cttattgtta aggcaatgca aggtctccta aaagatggaa acccgattcc ctcggccatc
     3661 gcggccaact ccggcatcta cggaggtgga ggtggatctc gtacgggtta gtaatgagtt
     3721 taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     3781 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     3841 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     3901 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     3961 tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc
     4021 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     4081 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     4141 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     4201 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtaccta gaagttccta
     4261 ttccgaagtt cctattctct agaaagtata ggaacttcct tggccaaaaa gcctgaactc
     4321 accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc cgacctgatg
     4381 cagctctcgg agggcgaaga atctcgtgct ttcagcttcg atgtaggagg gcgtggatat
     4441 gtcctgcggg taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac
     4501 tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca ttggggaatt cagcgagagc
     4561 ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc
     4621 gaactgcccg ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat
     4681 cttagccaga cgagcgggtt cggcccattc ggaccgcaag gaatcggtca atacactaca
     4741 tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt atcactggca aactgtgatg
     4801 gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct ttgggccgag
     4861 gactgccccg aagtccggca cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg
     4921 gacaatggcc gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa
     4981 tacgaggtcg ccaacatctt cttctggagg ccgtggttgg cttgtatgga gcagcagacg
     5041 cgctacttcg agcggaggca tccggagctt gcaggatcgc cgcggctccg ggcgtatatg
     5101 ctccgcattg gtcttgacca actctatcag agcttggttg acggcaattt cgatgatgca
     5161 gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt
     5221 acacaaatcg cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc
     5281 gatagtggaa accgacgccc cagcactcgt ccgagggcaa aggaatagca cgtactacga
     5341 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     5401 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
     5461 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     5521 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     5581 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
     5641 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     5701 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     5761 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     5821 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     5881 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     5941 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     6001 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     6061 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     6121 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     6181 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     6241 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     6301 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     6361 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     6421 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     6481 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     6541 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     6601 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     6661 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     6721 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     6781 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     6841 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     6901 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     6961 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     7021 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     7081 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     7141 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     7201 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     7261 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     7321 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     7381 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     7441 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     7501 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     7561 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     7621 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     7681 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     7741 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat
     7801 ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     7861 agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag
     7921 ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt
     7981 ttgcgctgct tcgcgatgta cgggccagat a
//
