LOCUS       sequence-376017-        8411 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 8411)
  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 8411)
  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: PRPF3
FEATURES             Location/Qualifiers
     source          1..8411
                     /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(3368..3392)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3517..3558
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(4013..4030)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4019..4243
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4376..4395)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4527..4574
                     /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             4582..5604
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4582..5604
                     /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    5734..5855
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5771..5790)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5825..5844
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5904..5920)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5904..5920)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5917..5939)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5928..5944
                     /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(5952..5958,5959..5976,5977..5982))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5997..6018
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6135..6152)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6306..6894)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6386..6405)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7065..7856,7857..7925))
                     /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     7688..7707
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7926..8030)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8105..8124)
                     /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 ggtacaaaaa agttggcatg
     1321 gcactgtcaa agagggagct ggatgagctg aaaccatgga tagagaagac agtgaagagg
     1381 gtcctgggtt tctcagagcc tacggtggtc acagcagcat tgaactgtgt ggggaagggc
     1441 atggacaaga agaaggcagc cgatcatctg aaaccttttc ttgatgattc tactctccga
     1501 tttgtggaca aactgtttga ggctgtggag gaaggccgaa gctctaggca ttccaagtct
     1561 agcagtgaca ggagcagaaa acgagagcta aaggaggtgt ttggtgatga ctctgagatc
     1621 tctaaagaat catcaggagt aaagaagcga cgaatacccc gttttgagga ggtggaagaa
     1681 gagccagagg tgatccctgg gcctccatca gagagccctg gcatgctgac taagctccag
     1741 atcaaacaga tgatggaggc agcaacacga caaatcgagg agaggaaaaa acagctgagc
     1801 ttcattagcc cccctacacc tcagccaaag actccttctt cctcccaacc agaacgactt
     1861 cctattggca acactattca gccctcccag gctgccactt tcatgaatga tgccattgag
     1921 aaggcaagga aagcagctga actgcaagct cgaatccaag cccagctggc actgaagcca
     1981 ggactcatcg gcaatgccaa catggtgggc ctggctaatc tccatgccat gggcattgct
     2041 cccccgaagg tggagttaaa agaccaaacg aaacctacac cactgatcct ggatgagcaa
     2101 gggcgcactg tagatgcaac aggcaaggag attgagctga cacaccgcat gcctactctg
     2161 aaagccaata ttcgtgctgt gaagagggaa caattcaagc aacaactaaa ggaaaagcca
     2221 tcagaagaca tggaatccaa tacctttttt gacccccgag tctccattgc cccttcccag
     2281 cgccagagac gcacttttaa attccatgac aagggcaaat ttgagaagat tgctcagcga
     2341 ttacggacaa aggctcaact ggagaagcta caggcagaga tttcacaagc agctcgaaaa
     2401 acaggcatcc atacttcgac taggcttgcc ctcattgctc ctaagaagga gctaaaggaa
     2461 ggagatattc ctgaaattga gtggtgggac tcttacataa tccccaatgg ctttgatctt
     2521 acagaggaaa atcccaagag agaagattat tttggaatca caaatcttgt tgaacatcca
     2581 gcccagctca atcctccagt tgacaatgac acaccagtta ctctgggagt atatcttacc
     2641 aagaaggaac agaaaaaact tcggagacaa acaaggaggg aagcacagaa ggaactacaa
     2701 gaaaaagtca ggctgggcct gatgcctcct ccagaaccca aagtgagaat ttctaatttg
     2761 atgcgagtat taggaacaga agctgttcaa gaccccacga aggtagaagc ccacgtcaga
     2821 gctcagatgg caaaaagaca gaaagcgcat gaagaggcca acgctgcccg aaaactcaca
     2881 gcagaacaga gaaaggtcaa gaaaattaaa aagcttaaag aagacatttc acagggggta
     2941 cacatatctg tatatagagt tcgaaatttg agcaacccag ccaagaagtt caagattgaa
     3001 gccaatgctg ggcaactgta cctgacaggg gtggtggtac tgcacaagga tgtcaacgtg
     3061 gtagtagtgg aagggggccc caaggcccag aagaaattta agcgtcttat gctgcatcgg
     3121 ataaagtggg atgaacagac atctaacaca aagggagatg atgatgagga gtctgatgag
     3181 gaagctgtga agaaaaccaa caaatgtgta ctagtctggg agggtacagc caaagaccgg
     3241 agctttggag agatgaagtt taaacagtgt cctacagaga acatggctcg tgagcatttc
     3301 aaaaagcatg gggctgaaca ctactgggac cttgcgctga gtgaatctgt gttagagtcc
     3361 actgattgcc caactttctt gtacaaagtg gttgatatcc agcacagtgg cggccgctcg
     3421 agtctagagg gcccgcggtt cgaaaccagc ggaggcggcg gaagcggggg aggaggcagc
     3481 ggcggaggcg gcagcggggg cggcggaagc gctaccggta agcctatccc taaccctctc
     3541 ctcggtctcg attctacgcg taccggtatg gcttctaact ttactcagtt cgttctcgtc
     3601 gacaatggcg gaactggcga cgtgactgtc gccccaagca acttcgctaa cggggtcgct
     3661 gaatggatca gctctaactc gcgatcacag gcttacaaag taacctgtag cgttcgtcag
     3721 agctctgcgc agaatcgcaa atacaccatc aaagtcgagg tgcctaaagt ggcaacccag
     3781 actgttggtg gtgaagagct tcctgtagcc ggatggagat cttacttaaa tatggaacta
     3841 accattccaa ttttcgccac gaattccgac tgcgagctta ttgttaaggc aatgcaaggt
     3901 ctcctaaaag atggaaaccc gattccctcg gccatcgcgg ccaactccgg catctacgga
     3961 ggtggaggtg gatctcgtac gggttagtaa tgagtttaaa cccgctgatc agcctcgact
     4021 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     4081 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     4141 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     4201 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     4261 accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg
     4321 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     4381 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     4441 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     4501 gattagggtg atggttcacg tacctagaag ttcctattcc gaagttccta ttctctagaa
     4561 agtataggaa cttccttggc caaaaagcct gaactcaccg cgacgtctgt cgagaagttt
     4621 ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct
     4681 cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc
     4741 gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt
     4801 ccggaagtgc ttgacattgg ggaattcagc gagagcctga cctattgcat ctcccgccgt
     4861 gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctgcagccg
     4921 gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc
     4981 ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt
     5041 gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc
     5101 gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc
     5161 gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc
     5221 attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc
     5281 tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg
     5341 gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct tgaccaactc
     5401 tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac
     5461 gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg
     5521 gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc
     5581 actcgtccga gggcaaagga atagcacgta ctacgagatt tcgattccac cgccgccttc
     5641 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     5701 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
     5761 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     5821 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     5881 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     5941 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     6001 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     6061 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     6121 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     6181 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     6241 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     6301 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     6361 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     6421 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     6481 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     6541 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     6601 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     6661 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     6721 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     6781 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     6841 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     6901 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     6961 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     7021 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     7081 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     7141 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     7201 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     7261 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     7321 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     7381 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     7441 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     7501 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     7561 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     7621 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     7681 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     7741 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     7801 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     7861 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     7921 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     7981 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     8041 cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc
     8101 tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt
     8161 tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg
     8221 acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg
     8281 ccagatatac gcgttgacat tgattattga ctaggctttt gcaaaaagct ttgcaaagat
     8341 ggataaagtt ttaaacagag aggaatcttt gcagctaatg gaccttctag gtcttgaaag
     8401 gagtgggaat t
//
