LOCUS       sequence-377207-        7927 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 7927)
  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 7927)
  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: PRPF4-1
FEATURES             Location/Qualifiers
     source          1..7927
                     /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"
     protein_bind    complement(2884..2908)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3033..3074
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3529..3546)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3535..3759
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3892..3911)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4043..4090
                     /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             4098..5120
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4098..5120
                     /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    5250..5371
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5287..5306)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5341..5360
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5420..5436)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5420..5436)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5433..5455)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5444..5460
                     /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(5468..5474,5475..5492,5493..5498))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5513..5534
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5651..5668)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5822..6410)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5902..5921)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6581..7372,7373..7441))
                     /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     7204..7223
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7442..7546)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7621..7640)
                     /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 gttggcatgg
     1321 cttcctcgcg agcctcttcc acgcaggcaa ccaaaactaa agcacccgac gacttagttg
     1381 ctccggtcgt gaagaaacca cacatctatt atggaagttt ggaagagaag gagagggagc
     1441 gtctggccaa aggagagtct gggattttgg ggaaagacgg acttaaagca gggatcgaag
     1501 ctggaaatat taatataacc tctggagaag tgtttgaaat tgaagagcat atcagcgagc
     1561 gacaggcaga agtattggct gagtttgaga gaaggaagcg agcccggcag atcaatgttt
     1621 ccacagatga ctcagaggtc aaagcttgcc ttagagcctt gggggaaccc atcacacttt
     1681 ttggagaggg tcctgctgaa agaagagaaa ggttaagaaa tatcctctca gttgtcggta
     1741 ctgatgcctt gaaaaagacc aaaaaggatg atgagaagtc taaaaagtcc aaagaagagt
     1801 atcagcaaac ctggtatcat gaaggaccaa atagcttgaa ggtggcaaga ctatggattg
     1861 ctaattattc gttgcccagg gcaatgaaac gcttggaaga ggcccgactc cataaggaga
     1921 ttcctgagac aacaaggacc tcccagatgc aagagctgca caagtctctc cggtctttga
     1981 ataatttttg cagtcagatt ggggatgatc ggcctatctc ctactgtcac tttagtccca
     2041 attccaagat gctggccaca gcttgttgga gtgggctttg caagctctgg tctgttcctg
     2101 attgcaacct ccttcacact cttcgagggc ataacacaaa tgtaggagca attgtattcc
     2161 atcccaaatc cactgtctcc ttggacccaa aagatgtcaa cctggcctct tgtgcggctg
     2221 atggctctgt gaagctttgg agtctcgaca gtgatgaacc agtggcagat attgaaggcc
     2281 atacagtgcg tgtggcgcgg gtaatgtggc atccttcagg acgtttcctg ggcaccacct
     2341 gctatgaccg ttcatggcgc ttatgggatt tggaggctca agaggagatc ctgcatcagg
     2401 aaggccatag catgggtgtg tatgacattg ccttccatca agatggctct ttggctggca
     2461 ctgggggact ggatgcattt ggtcgagttt gggacctacg cacaggacgt tgtatcatgt
     2521 tcttagaagg ccacctgaaa gaaatctatg gaataaattt ctcccccaat ggctatcaca
     2581 ttgcaaccgg cagtggtgac aacacctgca aagtgtggga cctccgacag cggcgttgcg
     2641 tctacaccat ccctgctcat cagaacttag tgactggtgt caagtttgag cctatccatg
     2701 ggaacttctt gcttactggt gcctatgata acacagccaa gatctggacg cacccaggct
     2761 ggtccccgct gaagactctg gctggccacg aaggcaaagt gatgggccta gatatttctt
     2821 ccgatgggca gctcatagcc acttgctcat atgacaggac cttcaagctg tggatggctg
     2881 aatacccaac tttcttgtac aaagtggttg atatccagca cagtggcggc cgctcgagtc
     2941 tagagggccc gcggttcgaa accagcggag gcggcggaag cgggggagga ggcagcggcg
     3001 gaggcggcag cgggggcggc ggaagcgcta ccggtaagcc tatccctaac cctctcctcg
     3061 gtctcgattc tacgcgtacc ggtatggctt ctaactttac tcagttcgtt ctcgtcgaca
     3121 atggcggaac tggcgacgtg actgtcgccc caagcaactt cgctaacggg gtcgctgaat
     3181 ggatcagctc taactcgcga tcacaggctt acaaagtaac ctgtagcgtt cgtcagagct
     3241 ctgcgcagaa tcgcaaatac accatcaaag tcgaggtgcc taaagtggca acccagactg
     3301 ttggtggtga agagcttcct gtagccggat ggagatctta cttaaatatg gaactaacca
     3361 ttccaatttt cgccacgaat tccgactgcg agcttattgt taaggcaatg caaggtctcc
     3421 taaaagatgg aaacccgatt ccctcggcca tcgcggccaa ctccggcatc tacggaggtg
     3481 gaggtggatc tcgtacgggt tagtaatgag tttaaacccg ctgatcagcc tcgactgtgc
     3541 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     3601 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     3661 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     3721 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca
     3781 gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     3841 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     3901 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     3961 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     4021 agggtgatgg ttcacgtacc tagaagttcc tattccgaag ttcctattct ctagaaagta
     4081 taggaacttc cttggccaaa aagcctgaac tcaccgcgac gtctgtcgag aagtttctga
     4141 tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa gaatctcgtg
     4201 ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg
     4261 gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg
     4321 aagtgcttga cattggggaa ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac
     4381 agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg
     4441 cggaggccat ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat
     4501 tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg
     4561 atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc
     4621 aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc
     4681 acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg
     4741 actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga
     4801 ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc
     4861 ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc
     4921 agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa
     4981 tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg
     5041 tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc
     5101 gtccgagggc aaaggaatag cacgtactac gagatttcga ttccaccgcc gccttctatg
     5161 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     5221 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     5281 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     5341 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     5401 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     5461 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     5521 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     5581 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     5641 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     5701 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     5761 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     5821 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     5881 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     5941 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     6001 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     6061 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     6121 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     6181 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     6241 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     6301 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     6361 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     6421 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     6481 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     6541 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     6601 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     6661 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     6721 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     6781 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     6841 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     6901 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     6961 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     7021 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     7081 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     7141 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     7201 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     7261 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     7321 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     7381 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     7441 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     7501 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     7561 aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag
     7621 tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga
     7681 ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa
     7741 ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag
     7801 atatacgcgt tgacattgat tattgactag gcttttgcaa aaagctttgc aaagatggat
     7861 aaagttttaa acagagagga atctttgcag ctaatggacc ttctaggtct tgaaaggagt
     7921 gggaatt
//
