LOCUS       sequence-375983-        7876 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 7876)
  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 7876)
  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: PLRG1
FEATURES             Location/Qualifiers
     source          1..7876
                     /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(2833..2857)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2982..3023
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3478..3495)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3484..3708
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3841..3860)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3992..4039
                     /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             4047..5069
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4047..5069
                     /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    5199..5320
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5236..5255)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5290..5309
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5369..5385)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5369..5385)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5382..5404)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5393..5409
                     /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(5417..5423,5424..5441,5442..5447))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5462..5483
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5600..5617)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5771..6359)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5851..5870)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6530..7321,7322..7390))
                     /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     7153..7172
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7391..7495)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7570..7589)
                     /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 tcgaggaggt acagaaacat tctgtacaca cccttgtgtt caggtcgttg aagaggaccc
     1381 atgacatgtt tgtagctgat aatggaaaac ctgtgccttt agatgaagag agtcacaaac
     1441 gaaaaatggc aatcaagctt cgtaatgagt atggtcctgt gttgcatatg cctacttcaa
     1501 aagaaaatct taaagagaag ggtcctcaga atgcaacgga ttcatatgtt cataaacagt
     1561 accctgccaa tcaaggacaa gaagttgaat actttgtggc aggggttgct ttgacagcag
     1621 atactaagat ccagagaatg ccaagtgaat cagctgcaca gtccttagcg gtggcattac
     1681 ctttgcagac caaggctgat gcaaatcgta ctgcccctag tggaagtgaa taccgacatc
     1741 ctggggcttc tgaccgtcca cagcctacag cgatgaattc aattgtcatg gagactggca
     1801 ataccaagaa ctctgcactg atggctaaaa aagcccctac aatgccaaaa ccccagtggc
     1861 acccaccgtg gaaactctac agggttatca gtgggcatct tggctgggtt cgatgtattg
     1921 ctgtggaacc tggaaatcag tggtttgtta ctggatctgc tgacagaact ataaagatct
     1981 gggacttggc tagtggcaaa ttaaaactgt cattgactgg gcatattagt actgtgcggg
     2041 gcgtgatagt aagcacaagg agcccatatc tgttctcttg tggagaagac aaacaagtga
     2101 aatgctggga tctcgaatac aataaggtta tacggcatta tcatggacat ttaagtgcag
     2161 tgtatggttt ggatttgcac ccgacaatcg atgtgttggt aacctgtagt cgagattcaa
     2221 ctgcacggat ttgggatgtg agaactaaag ccagtgtaca cacattatct ggacatacaa
     2281 atgcagttgc tacagtgaga tgtcaggctg cagaaccaca gattattaca ggaagccatg
     2341 atactacaat tcgattatgg gatctggtgg ctggaaaaac aagagtgaca ttaacaaatc
     2401 acaaaaaatc agttagggct gtggttttac atccaagaca ttacacattt gcatctggtt
     2461 ctccagataa cataaagcag tggaaattcc ctgatggaag tttcatacaa aatctttccg
     2521 gtcataatgc tattattaac acattgacgg taaattctga tggagtgctt gtatctggag
     2581 ctgacaatgg caccatgcat ctttgggact ggagaactgg ctacaatttt cagagagttc
     2641 acgcagctgt gcaacctggg tctttggaca gtgaatcagg aatatttgct tgtgcttttg
     2701 atcagtctga aagtcgatta ctaacagctg aagctgataa aaccattaaa gtatacagag
     2761 aggatgacac agccacagaa gaaactcatc cagtcagctg gaaaccagaa attatcaaga
     2821 gaaagagatt ttacccaact ttcttgtaca aagtggttga tatccagcac agtggcggcc
     2881 gctcgagtct agagggcccg cggttcgaaa ccagcggagg cggcggaagc gggggaggag
     2941 gcagcggcgg aggcggcagc gggggcggcg gaagcgctac cggtaagcct atccctaacc
     3001 ctctcctcgg tctcgattct acgcgtaccg gtatggcttc taactttact cagttcgttc
     3061 tcgtcgacaa tggcggaact ggcgacgtga ctgtcgcccc aagcaacttc gctaacgggg
     3121 tcgctgaatg gatcagctct aactcgcgat cacaggctta caaagtaacc tgtagcgttc
     3181 gtcagagctc tgcgcagaat cgcaaataca ccatcaaagt cgaggtgcct aaagtggcaa
     3241 cccagactgt tggtggtgaa gagcttcctg tagccggatg gagatcttac ttaaatatgg
     3301 aactaaccat tccaattttc gccacgaatt ccgactgcga gcttattgtt aaggcaatgc
     3361 aaggtctcct aaaagatgga aacccgattc cctcggccat cgcggccaac tccggcatct
     3421 acggaggtgg aggtggatct cgtacgggtt agtaatgagt ttaaacccgc tgatcagcct
     3481 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     3541 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     3601 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     3661 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg
     3721 aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg
     3781 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     3841 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     3901 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     3961 aacttgatta gggtgatggt tcacgtacct agaagttcct attccgaagt tcctattctc
     4021 tagaaagtat aggaacttcc ttggccaaaa agcctgaact caccgcgacg tctgtcgaga
     4081 agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag
     4141 aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct
     4201 gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc
     4261 cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc
     4321 gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc
     4381 agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt
     4441 tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg
     4501 cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt
     4561 ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc
     4621 acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag
     4681 cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct
     4741 tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc
     4801 atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc
     4861 aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat
     4921 gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa
     4981 gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc
     5041 ccagcactcg tccgagggca aaggaatagc acgtactacg agatttcgat tccaccgccg
     5101 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     5161 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     5221 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     5281 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     5341 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     5401 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     5461 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5521 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5581 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     5641 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     5701 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5761 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     5821 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     5881 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     5941 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     6001 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     6061 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     6121 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     6181 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     6241 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     6301 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     6361 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     6421 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     6481 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6541 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6601 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6661 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     6721 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     6781 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     6841 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     6901 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     6961 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     7021 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     7081 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     7141 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     7201 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     7261 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     7321 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     7381 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     7441 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     7501 ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg
     7561 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg
     7621 tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt
     7681 gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt
     7741 acgggccaga tatacgcgtt gacattgatt attgactagg cttttgcaaa aagctttgca
     7801 aagatggata aagttttaaa cagagaggaa tctttgcagc taatggacct tctaggtctt
     7861 gaaaggagtg ggaatt
//
