LOCUS       sequence-377216-        7885 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 7885)
  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 7885)
  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: EDC3
FEATURES             Location/Qualifiers
     source          1..7885
                     /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    complement(2842..2866)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2991..3032
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3487..3504)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3493..3717
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3850..3869)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4001..4048
                     /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             4056..5078
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4056..5078
                     /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    5208..5329
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5245..5264)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5299..5318
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5378..5394)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5378..5394)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5391..5413)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5402..5418
                     /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(5426..5432,5433..5450,5451..5456))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5471..5492
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5609..5626)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5780..6368)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5860..5879)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6539..7330,7331..7399))
                     /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     7162..7181
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7400..7504)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7579..7598)
                     /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 gtgcaaaaaa gttggcatgg
     1321 ctacagattg gctgggaagt attgtgtcca tcaattgtgg agatagcttg ggtgtctatc
     1381 agggaagagt gtcagctgtg gatcaggtca gccagaccat ttctctcacc cggcctttcc
     1441 ataatggagt gaagtgtctt gttccagaag tcaccttcag ggcaggtgac attacggagt
     1501 taaaaattct ggagatacca ggacctggag acaaccaaca ttttggagac cttcatcaaa
     1561 cagaattagg cccctctggt gctggctgcc aagtgggcat caatcagaat ggcacaggca
     1621 agtttgtcaa gaagccagcc tcttccagca gtgcccctca gaatatccct aagaggacag
     1681 atgtgaagag ccaggatgtt gccgtttccc cgcagcagca acagtgctca aagagctatg
     1741 tcgacaggca catggaatcc ttgagtcagt ccaaaagttt ccgtcgtcgg cacaactcct
     1801 ggtcatctag tagcaggcac ccaaatcagg caactcccaa gaaaagtggt ttaaagaatg
     1861 gccagatgaa gaataaagat gacgagtgct tcggggatga tattgaggag atcccagaca
     1921 cagattttga ttttgaaggg aacctggctc tttttgacaa ggcagctgtg tttgaggaga
     1981 ttgataccta tgaaaggaga agtggtaccc gttcccgggg catcccaaat gaaaggccca
     2041 ctcggtaccg ccatgatgag aacatcttgg agtccgagcc cattgtctat cgacggatca
     2101 tagtgcccca caacgtgagc aaggagttct gcacggactc tggcctggtt gtcccaagta
     2161 tttcctatga gctgcataaa aagctgttgt ccgtggctga gaagcatggg ctgacccttg
     2221 agcggagact ggagatgaca ggtgtgtgtg ccagtcagat ggcactgacc ctcctcggag
     2281 gacctaacag gttgaatccc aaaaatgttc accagaggcc tacagtggct ctactgtgtg
     2341 gacctcatgt gaagggggct cagggtatca gctgtggaag gcacctagcc aaccatgatg
     2401 tccaggtcat ccttttcctg cccaattttg tcaagatgtt ggaatctatc accaatgagc
     2461 tgtcgctctt cagcaagacc caaggccaac aagtgtctag cctcaaagat ctgcccacta
     2521 gccctgtgga cctggtcatc aactgcctgg attgccctga gaacgtcttc ctgcgcgatc
     2581 aaccctggta caaggcagct gtggcctggg ccaaccagaa ccgggcacca gtactcagca
     2641 tagaccctcc tgtgcatgaa gtcgaacagg gcattgatgc caaatggtca ctggcactgg
     2701 gcctgcctct gccactgggg gagcacgcag gccgtatcta tttgtgcgac attggcattc
     2761 cccagcaggt cttccaggag gtgggcatca actaccactc gccctttggc tgcaagtttg
     2821 ttatcccact gcactctgct tacccaactt tcttgtacaa agtggttgat atccagcaca
     2881 gtggcggccg ctcgagtcta gagggcccgc ggttcgaaac cagcggaggc ggcggaagcg
     2941 ggggaggagg cagcggcgga ggcggcagcg ggggcggcgg aagcgctacc ggtaagccta
     3001 tccctaaccc tctcctcggt ctcgattcta cgcgtaccgg tatggcttct aactttactc
     3061 agttcgttct cgtcgacaat ggcggaactg gcgacgtgac tgtcgcccca agcaacttcg
     3121 ctaacggggt cgctgaatgg atcagctcta actcgcgatc acaggcttac aaagtaacct
     3181 gtagcgttcg tcagagctct gcgcagaatc gcaaatacac catcaaagtc gaggtgccta
     3241 aagtggcaac ccagactgtt ggtggtgaag agcttcctgt agccggatgg agatcttact
     3301 taaatatgga actaaccatt ccaattttcg ccacgaattc cgactgcgag cttattgtta
     3361 aggcaatgca aggtctccta aaagatggaa acccgattcc ctcggccatc gcggccaact
     3421 ccggcatcta cggaggtgga ggtggatctc gtacgggtta gtaatgagtt taaacccgct
     3481 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     3541 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     3601 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     3661 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt
     3721 ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg
     3781 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     3841 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     3901 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     3961 accccaaaaa acttgattag ggtgatggtt cacgtaccta gaagttccta ttccgaagtt
     4021 cctattctct agaaagtata ggaacttcct tggccaaaaa gcctgaactc accgcgacgt
     4081 ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc cgacctgatg cagctctcgg
     4141 agggcgaaga atctcgtgct ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg
     4201 taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac tttgcatcgg
     4261 ccgcgctccc gattccggaa gtgcttgaca ttggggaatt cagcgagagc ctgacctatt
     4321 gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg
     4381 ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat cttagccaga
     4441 cgagcgggtt cggcccattc ggaccgcaag gaatcggtca atacactaca tggcgtgatt
     4501 tcatatgcgc gattgctgat ccccatgtgt atcactggca aactgtgatg gacgacaccg
     4561 tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct ttgggccgag gactgccccg
     4621 aagtccggca cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg gacaatggcc
     4681 gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa tacgaggtcg
     4741 ccaacatctt cttctggagg ccgtggttgg cttgtatgga gcagcagacg cgctacttcg
     4801 agcggaggca tccggagctt gcaggatcgc cgcggctccg ggcgtatatg ctccgcattg
     4861 gtcttgacca actctatcag agcttggttg acggcaattt cgatgatgca gcttgggcgc
     4921 agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt acacaaatcg
     4981 cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc gatagtggaa
     5041 accgacgccc cagcactcgt ccgagggcaa aggaatagca cgtactacga gatttcgatt
     5101 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     5161 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     5221 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     5281 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     5341 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     5401 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     5461 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     5521 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     5581 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     5641 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     5701 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     5761 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5821 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5881 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5941 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     6001 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     6061 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     6121 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     6181 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     6241 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     6301 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     6361 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     6421 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     6481 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     6541 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     6601 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     6661 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     6721 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     6781 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     6841 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6901 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6961 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     7021 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     7081 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     7141 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     7201 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     7261 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     7321 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     7381 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     7441 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     7501 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc
     7561 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
     7621 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
     7681 gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
     7741 tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactaggc ttttgcaaaa
     7801 agctttgcaa agatggataa agttttaaac agagaggaat ctttgcagct aatggacctt
     7861 ctaggtcttg aaaggagtgg gaatt
//
