LOCUS       sequence-376711-        7855 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 7855)
  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 7855)
  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: CLK2
FEATURES             Location/Qualifiers
     source          1..7855
                     /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(2812..2836)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2961..3002
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3457..3474)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3463..3687
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3820..3839)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3971..4018
                     /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             4026..5048
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4026..5048
                     /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    5178..5299
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5215..5234)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5269..5288
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5348..5364)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5348..5364)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5361..5383)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5372..5388
                     /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(5396..5402,5403..5420,5421..5426))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5441..5462
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5579..5596)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5750..6338)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5830..5849)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6509..7300,7301..7369))
                     /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     7132..7151
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7370..7474)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7549..7568)
                     /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 gttggcatgc
     1321 cgcatcctcg aaggtaccac tcctcagagc gaggcagccg ggggagttac cgtgaacact
     1381 atcggagccg aaagcataag cgacgaagaa gtcgctcctg gtcaagtagt agtgaccgga
     1441 cacgacggcg tcggcgagag gacagctacc atgtccgttc tcgaagcagt tatgatgatc
     1501 gttcgtccga ccggagggtg tatgaccggc gatactgtgg cagctacaga cgcaacgatt
     1561 atagccggga tcggggagat gcctactatg acacagacta tcggcattcc tatgaatatc
     1621 agcgggagaa cagcagttac cgcagccagc gcagcagccg gaggaagcac agacggcgga
     1681 ggaggcgcag ccggacattt agccgctcat cttcgcacag cagccggaga gccaagagtg
     1741 tagaggacga cgctgagggc cacctcatct accacgtcgg ggactggcta caagagcgat
     1801 atgaaatcgt tagcacctta ggagagggga ccttcggccg agttgtacaa tgtgttgacc
     1861 atcgcagggg tggggctcga gttgccctga agatcattaa gaatgtggag aagtacaagg
     1921 aagcagctcg acttgagatc aacgtgctag agaaaatcaa tgagaaagac cctgacaaca
     1981 agaacctctg tgtccagatg tttgactggt ttgactacca tggccacatg tgtatctcct
     2041 ttgagcttct gggccttagc accttcgatt tcctcaaaga caacaactac ctgccctacc
     2101 ccatccacca agtgcgccac atggccttcc agctgtgcca ggctgtcaag ttcctccatg
     2161 ataacaagct gacacataca gacctcaagc ctgaaaatat tctgtttgtg aattcagact
     2221 atgagctcac ctacaaccta gagaagaagc gagatgagcg cagtgtgaag agcacagctg
     2281 tgcgggtggt agactttggc agtgccacct ttgaccatga gcaccatagc accattgtct
     2341 ccactcgcca ttaccgagca ccagaagtca tccttgagtt gggctggtca cagccttgtg
     2401 atgtgtggag tataggctgc atcatctttg aatactatgt gggattcacc ctcttccaga
     2461 cccatgacaa cagagagcat ctagccatga tggaaaggat cttgggtcct atcccttccc
     2521 ggatgatccg aaagacaaga aagcagaaat atttttaccg gggtcgcctg gattgggatg
     2581 agaacacatc agctgggcgc tatgttcgtg agaactgcaa accgctgcgg cggtatctga
     2641 cctcagaggc agaggaacac caccagctct tcgatctgat tgaaagcatg ctagagtatg
     2701 aaccagctaa gcggctgacc ttgggtgaag cccttcagca tcctttcttc gcccgccttc
     2761 gggctgagcc gcccaacaag ttgtgggact ccagtcggga tatcagtcgg tgcccaactt
     2821 tcttgtacaa agtggttgat atccagcaca gtggcggccg ctcgagtcta gagggcccgc
     2881 ggttcgaaac cagcggaggc ggcggaagcg ggggaggagg cagcggcgga ggcggcagcg
     2941 ggggcggcgg aagcgctacc ggtaagccta tccctaaccc tctcctcggt ctcgattcta
     3001 cgcgtaccgg tatggcttct aactttactc agttcgttct cgtcgacaat ggcggaactg
     3061 gcgacgtgac tgtcgcccca agcaacttcg ctaacggggt cgctgaatgg atcagctcta
     3121 actcgcgatc acaggcttac aaagtaacct gtagcgttcg tcagagctct gcgcagaatc
     3181 gcaaatacac catcaaagtc gaggtgccta aagtggcaac ccagactgtt ggtggtgaag
     3241 agcttcctgt agccggatgg agatcttact taaatatgga actaaccatt ccaattttcg
     3301 ccacgaattc cgactgcgag cttattgtta aggcaatgca aggtctccta aaagatggaa
     3361 acccgattcc ctcggccatc gcggccaact ccggcatcta cggaggtgga ggtggatctc
     3421 gtacgggtta gtaatgagtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
     3481 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     3541 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     3601 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     3661 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta
     3721 gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     3781 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     3841 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     3901 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     3961 cacgtaccta gaagttccta ttccgaagtt cctattctct agaaagtata ggaacttcct
     4021 tggccaaaaa gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg
     4081 acagcgtctc cgacctgatg cagctctcgg agggcgaaga atctcgtgct ttcagcttcg
     4141 atgtaggagg gcgtggatat gtcctgcggg taaatagctg cgccgatggt ttctacaaag
     4201 atcgttatgt ttatcggcac tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca
     4261 ttggggaatt cagcgagagc ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt
     4321 tgcaagacct gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg gaggccatgg
     4381 atgcgatcgc tgcggccgat cttagccaga cgagcgggtt cggcccattc ggaccgcaag
     4441 gaatcggtca atacactaca tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt
     4501 atcactggca aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg
     4561 agctgatgct ttgggccgag gactgccccg aagtccggca cctcgtgcac gcggatttcg
     4621 gctccaacaa tgtcctgacg gacaatggcc gcataacagc ggtcattgac tggagcgagg
     4681 cgatgttcgg ggattcccaa tacgaggtcg ccaacatctt cttctggagg ccgtggttgg
     4741 cttgtatgga gcagcagacg cgctacttcg agcggaggca tccggagctt gcaggatcgc
     4801 cgcggctccg ggcgtatatg ctccgcattg gtcttgacca actctatcag agcttggttg
     4861 acggcaattt cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg
     4921 gagccgggac tgtcgggcgt acacaaatcg cccgcagaag cgcggccgtc tggaccgatg
     4981 gctgtgtaga agtactcgcc gatagtggaa accgacgccc cagcactcgt ccgagggcaa
     5041 aggaatagca cgtactacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     5101 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     5161 agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     5221 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     5281 aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt
     5341 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     5401 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     5461 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     5521 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     5581 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     5641 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     5701 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     5761 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     5821 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     5881 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     5941 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     6001 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     6061 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     6121 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     6181 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     6241 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     6301 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     6361 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     6421 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     6481 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     6541 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     6601 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     6661 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     6721 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     6781 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     6841 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     6901 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     6961 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     7021 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     7081 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     7141 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     7201 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     7261 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     7321 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     7381 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     7441 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     7501 acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc
     7561 tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta
     7621 gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa
     7681 tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg
     7741 acattgatta ttgactaggc ttttgcaaaa agctttgcaa agatggataa agttttaaac
     7801 agagaggaat ctttgcagct aatggacctt ctaggtcttg aaaggagtgg gaatt
//
