LOCUS       sequence-376000-        7394 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 7394)
  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 7394)
  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: PRPF18
FEATURES             Location/Qualifiers
     source          1..7394
                     /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"
     regulatory      1318..1327
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(2351..2375)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2500..2541
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2996..3013)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3002..3226
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3359..3378)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3510..3557
                     /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             3565..4587
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3565..4587
                     /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    4717..4838
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4754..4773)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4808..4827
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4887..4903)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4887..4903)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4900..4922)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4911..4927
                     /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(4935..4941,4942..4959,4960..4965))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4980..5001
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5118..5135)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5289..5877)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5369..5388)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6048..6839,6840..6908))
                     /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     6671..6690
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6909..7013)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7088..7107)
                     /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 gcaggctgcc
     1321 accatggaca ttctgaaatc agagatcctt cggaagcggc agctggtgga ggacaggaac
     1381 ctgctggtgg aaaataaaaa atatttcaag cgtagtgagc tcgccaaaaa agaagaggaa
     1441 gcatattttg aaagatgtgg ctacaagata cagccaaaag aggaggacca gaaaccatta
     1501 acttcatcga atccagtgtt agaacttgaa ctggcagagg aaaaattacc tatgacgctt
     1561 tctaggcaag aggtcatcag aagattgaga gaaagaggag aaccaatcag actatttgga
     1621 gagactgatt atgatgcttt tcaacgttta aggaaaatag agatcctcac accagaagtt
     1681 aacaagggat tgaggaatga tttgaaagca gccttggata agattgatca gcagtacctc
     1741 aatgaaatcg tcggcggtca ggagcctgga gaggaagaca cacagaatga tctgaaagtt
     1801 catgaggaaa acaccacaat tgaagagtta gaggcgcttg gagagtcctt agggaaaggc
     1861 gatgatcata aggacatgga catcatcacc aaattcctga agtttcttct tggcgtttgg
     1921 gctaaagaat tgaatgccag agaagattat gtgaaacgca gtgtgcaggg taaactgaac
     1981 agtgcgaccc agaaacagac cgagtcctac ctaagaccac tttttagaaa gctacggaaa
     2041 aggaatcttc ctgctgatat taaagaatca ataacggata ttattaaatt catgttgcag
     2101 agagaatacg tgaaggcaaa tgatgcttat cttcagatgg ccattggaaa tgcgccttgg
     2161 cccatcggtg tcactatggt tggtatccat gccagaactg gcagagaaaa gattttttcc
     2221 aagcatgttg cacatgtttt aaatgacgaa actcagcgga aatatattca gggattgaag
     2281 aggttaatga ccatttgcca gaaacacttt cctacagacc catccaaatg tgtggagtac
     2341 aatgcactgg acccagcttt cttgtacaaa gtggttgata tccagcacag tggcggccgc
     2401 tcgagtctag agggcccgcg gttcgaaacc agcggaggcg gcggaagcgg gggaggaggc
     2461 agcggcggag gcggcagcgg gggcggcgga agcgctaccg gtaagcctat ccctaaccct
     2521 ctcctcggtc tcgattctac gcgtaccggt atggcttcta actttactca gttcgttctc
     2581 gtcgacaatg gcggaactgg cgacgtgact gtcgccccaa gcaacttcgc taacggggtc
     2641 gctgaatgga tcagctctaa ctcgcgatca caggcttaca aagtaacctg tagcgttcgt
     2701 cagagctctg cgcagaatcg caaatacacc atcaaagtcg aggtgcctaa agtggcaacc
     2761 cagactgttg gtggtgaaga gcttcctgta gccggatgga gatcttactt aaatatggaa
     2821 ctaaccattc caattttcgc cacgaattcc gactgcgagc ttattgttaa ggcaatgcaa
     2881 ggtctcctaa aagatggaaa cccgattccc tcggccatcg cggccaactc cggcatctac
     2941 ggaggtggag gtggatctcg tacgggttag taatgagttt aaacccgctg atcagcctcg
     3001 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     3061 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     3121 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     3181 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
     3241 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
     3301 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     3361 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     3421 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     3481 cttgattagg gtgatggttc acgtacctag aagttcctat tccgaagttc ctattctcta
     3541 gaaagtatag gaacttcctt ggccaaaaag cctgaactca ccgcgacgtc tgtcgagaag
     3601 tttctgatcg aaaagttcga cagcgtctcc gacctgatgc agctctcgga gggcgaagaa
     3661 tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc
     3721 gccgatggtt tctacaaaga tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg
     3781 attccggaag tgcttgacat tggggaattc agcgagagcc tgacctattg catctcccgc
     3841 cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc tgttctgcag
     3901 ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc ttagccagac gagcgggttc
     3961 ggcccattcg gaccgcaagg aatcggtcaa tacactacat ggcgtgattt catatgcgcg
     4021 attgctgatc cccatgtgta tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc
     4081 gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg actgccccga agtccggcac
     4141 ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg acaatggccg cataacagcg
     4201 gtcattgact ggagcgaggc gatgttcggg gattcccaat acgaggtcgc caacatcttc
     4261 ttctggaggc cgtggttggc ttgtatggag cagcagacgc gctacttcga gcggaggcat
     4321 ccggagcttg caggatcgcc gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa
     4381 ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgca gggtcgatgc
     4441 gacgcaatcg tccgatccgg agccgggact gtcgggcgta cacaaatcgc ccgcagaagc
     4501 gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc
     4561 agcactcgtc cgagggcaaa ggaatagcac gtactacgag atttcgattc caccgccgcc
     4621 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     4681 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat
     4741 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     4801 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc
     4861 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     4921 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     4981 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     5041 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     5101 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     5161 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     5221 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     5281 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     5341 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     5401 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     5461 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     5521 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     5581 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     5641 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     5701 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     5761 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     5821 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     5881 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     5941 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     6001 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     6061 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     6121 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     6181 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     6241 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     6301 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     6361 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     6421 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     6481 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     6541 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     6601 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     6661 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     6721 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     6781 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     6841 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     6901 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     6961 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     7021 ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca
     7081 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg
     7141 tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga
     7201 ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac
     7261 gggccagata tacgcgttga cattgattat tgactaggct tttgcaaaaa gctttgcaaa
     7321 gatggataaa gttttaaaca gagaggaatc tttgcagcta atggaccttc taggtcttga
     7381 aaggagtggg aatt
//
