LOCUS       sequence-376025-        7924 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 7924)
  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 7924)
  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
FEATURES             Location/Qualifiers
     source          1..7924
                     /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(2881..2905)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3030..3071
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3526..3543)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3532..3756
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3889..3908)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4040..4087
                     /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             4095..5117
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4095..5117
                     /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    5247..5368
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5284..5303)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5338..5357
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5417..5433)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5417..5433)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5430..5452)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5441..5457
                     /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(5465..5471,5472..5489,5490..5495))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5510..5531
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5648..5665)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5819..6407)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5899..5918)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6578..7369,7370..7438))
                     /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     7201..7220
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7439..7543)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7618..7637)
                     /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 acggcaacca aaactaaagc acccgacgac ttagttgctc
     1381 cggtcgtgaa gaaaccacac atctattatg gaagtttgga agagaaggag agggagcgtc
     1441 tggccaaagg agagtctggg attttgggga aagacggact taaagcaggg atcgaagctg
     1501 gaaatattaa tataacctct ggagaagtgt ttgaaattga agagcatatc agcgagcgac
     1561 aggcagaagt attggctgag tttgagagaa ggaagcgagc ccggcagatc aatgtttcca
     1621 cagatgactc agaggtcaaa gcttgcctta gagccttggg ggaacccatc acactttttg
     1681 gagagggtcc tgctgaaaga agagaaaggt taagaaatat cctctcagtt gtcggtactg
     1741 atgccttgaa aaagaccaaa aaggatgatg agaagtctaa aaagtccaaa gaagagtatc
     1801 agcaaacctg gtatcatgaa ggaccaaata gcttgaaggt ggcaagacta tggattgcta
     1861 attattcgtt gcccagggca atgaaacgct tggaagaggc ccgactccat aaggagattc
     1921 ctgagacaac aaggacctcc cagatgcaag agctgcacaa gtctctccgg tctttgaata
     1981 atttttgcag tcagattggg gatgatcggc ctatctccta ctgtcacttt agtcccaatt
     2041 ccaagatgct ggccacagct tgttggagtg ggctttgcaa gctctggtct gttcctgatt
     2101 gcaacctcct tcacactctt cgagggcata acacaaatgt aggagcaatt gtattccatc
     2161 ccaaatccac tgtctccttg gacccaaaag atgtcaacct ggcctcttgt gcggctgatg
     2221 gctctgtgaa gctttggagt ctcgacagtg atgaaccagt ggcagatatt gaaggccata
     2281 cagtgcgtgt ggcgcgggta atgtggcatc cttcaggacg tttcctgggc accacctgct
     2341 atgaccgttc atggcgctta tgggatttgg aggctcaaga ggagatcctg catcaggaag
     2401 gccatagcat gggtgtgtat gacattgcct tccatcaaga tggctctttg gctggcactg
     2461 ggggactgga tgcatttggt cgagtttggg acctacgcac aggacgttgt atcatgttct
     2521 tagaaggcca cctgaaagaa atctatggaa taaatttctc ccccaatggc tatcacattg
     2581 caaccggcag tggtgacaac acctgcaaag tgtgggacct ccgacagcgg cgttgcgtct
     2641 acaccatccc tgctcatcag aacttagtga ctggtgtcaa gtttgagcct atccatggga
     2701 acttcttgct tactggtgcc tatgataaca cagccaagat ctggacgcac ccaggctggt
     2761 ccccgctgaa gactctggct ggccacgaag gcaaagtgat gggcctagat atttcttccg
     2821 atgggcagct catagccact tgctcatatg acaggacctt caagctgtgg atggctgaat
     2881 acccaacttt cttgtacaaa gtggttgata tccagcacag tggcggccgc tcgagtctag
     2941 agggcccgcg gttcgaaacc agcggaggcg gcggaagcgg gggaggaggc agcggcggag
     3001 gcggcagcgg gggcggcgga agcgctaccg gtaagcctat ccctaaccct ctcctcggtc
     3061 tcgattctac gcgtaccggt atggcttcta actttactca gttcgttctc gtcgacaatg
     3121 gcggaactgg cgacgtgact gtcgccccaa gcaacttcgc taacggggtc gctgaatgga
     3181 tcagctctaa ctcgcgatca caggcttaca aagtaacctg tagcgttcgt cagagctctg
     3241 cgcagaatcg caaatacacc atcaaagtcg aggtgcctaa agtggcaacc cagactgttg
     3301 gtggtgaaga gcttcctgta gccggatgga gatcttactt aaatatggaa ctaaccattc
     3361 caattttcgc cacgaattcc gactgcgagc ttattgttaa ggcaatgcaa ggtctcctaa
     3421 aagatggaaa cccgattccc tcggccatcg cggccaactc cggcatctac ggaggtggag
     3481 gtggatctcg tacgggttag taatgagttt aaacccgctg atcagcctcg actgtgcctt
     3541 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     3601 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     3661 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     3721 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     3781 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     3841 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     3901 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     3961 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     4021 gtgatggttc acgtacctag aagttcctat tccgaagttc ctattctcta gaaagtatag
     4081 gaacttcctt ggccaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg
     4141 aaaagttcga cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt
     4201 tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt
     4261 tctacaaaga tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag
     4321 tgcttgacat tggggaattc agcgagagcc tgacctattg catctcccgc cgtgcacagg
     4381 gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg
     4441 aggccatgga tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg
     4501 gaccgcaagg aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc
     4561 cccatgtgta tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg
     4621 ctctcgatga gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg
     4681 cggatttcgg ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact
     4741 ggagcgaggc gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc
     4801 cgtggttggc ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg
     4861 caggatcgcc gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga
     4921 gcttggttga cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg
     4981 tccgatccgg agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct
     5041 ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc
     5101 cgagggcaaa ggaatagcac gtactacgag atttcgattc caccgccgcc ttctatgaaa
     5161 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     5221 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     5281 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     5341 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     5401 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     5461 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     5521 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     5581 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     5641 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     5701 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     5761 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     5821 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     5881 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     5941 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     6001 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     6061 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     6121 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     6181 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     6241 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     6301 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     6361 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     6421 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     6481 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     6541 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     6601 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     6661 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     6721 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     6781 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     6841 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     6901 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     6961 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     7021 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     7081 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     7141 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     7201 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     7261 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     7321 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     7381 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     7441 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     7501 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     7561 tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac
     7621 aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt
     7681 cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg
     7741 catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata
     7801 tacgcgttga cattgattat tgactaggct tttgcaaaaa gctttgcaaa gatggataaa
     7861 gttttaaaca gagaggaatc tttgcagcta atggaccttc taggtcttga aaggagtggg
     7921 aatt
//
