LOCUS       sequence-376057-        7813 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 7813)
  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 7813)
  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: CLK1
FEATURES             Location/Qualifiers
     source          1..7813
                     /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(2770..2794)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2919..2960
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3415..3432)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3421..3645
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3778..3797)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3929..3976
                     /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             3984..5006
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3984..5006
                     /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    5136..5257
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5173..5192)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5227..5246
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5306..5322)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5306..5322)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5319..5341)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5330..5346
                     /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(5354..5360,5361..5378,5379..5384))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5399..5420
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5537..5554)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5708..6296)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5788..5807)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6467..7258,7259..7327))
                     /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     7090..7109
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7328..7432)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7507..7526)
                     /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 gttggcatga
     1321 gacactcaaa gagaacttac tgtcctgatt gggatgacaa ggattgggat tatggaaaat
     1381 ggaggagcag cagcagtcat aaaagaagga agagatcaca tagcagtgcc caggagaaca
     1441 agcgctgcaa atacaatcac tctaaaatgt gtgatagcca ttatttggaa agcaggtcta
     1501 taaatgagaa agattatcat agtcgacgct acattgatga gtacagaaat gactacactc
     1561 aaggatgtga acctggacat cgccaaagag accatgaaag ccggtatcag aaccatagta
     1621 gcaagtcttc tggtagaagt ggaagaagta gttataaaag caaacacagg attcaccaca
     1681 gtacttcaca tcgtcgttca catgggaaga gtcaccgaag gaaaagaacc aggagtgtag
     1741 aggatgatga ggagggtcac ctgatctgtc agagtggaga cgtactaagt gcaagatatg
     1801 aaattgttga tactttaggt gaaggagctt ttggaaaagt tgtggagtgc atcgatcata
     1861 aagcgggagg tagacatgta gcagtaaaaa tagttaaaaa tgtggataga tactgtgaag
     1921 ctgctcgctc agaaatacaa gttctggaac atctgaatac aacagacccc aacagtactt
     1981 tccgctgtgt ccagatgttg gaatggtttg agcatcatgg tcacatttgc attgtttttg
     2041 aactattggg acttagtact tacgacttca ttaaagaaaa tggttttcta ccatttcgac
     2101 tggatcatat cagaaagatg gcatatcaga tatgcaagtc tgtgaatttt ttgcacagta
     2161 ataagttgac tcacacagac ttaaagcctg aaaacatctt atttgtgcag tctgactaca
     2221 cagaggcgta taatcccaaa ataaaacgtg atgaacgcac cttaataaat ccagatatta
     2281 aagttgtaga ctttggtagt gcaacatatg atgacgaaca tcacagtaca ttggtatcta
     2341 caagacatta tagagcacct gaagttattt tagccctagg gtggtcccaa ccatgtgatg
     2401 tctggagcat aggatgcatt cttattgaat actatcttgg gtttaccgta tttccaacac
     2461 acgatagtaa ggagcattta gcaatgatgg aaaggattct tggacctcta ccaaaacata
     2521 tgatacagaa aaccaggaaa cgtaaatatt ttcaccacga tcgattagac tgggatgaac
     2581 acagttctgc cggcagatat gtttcaagac gctgtaaacc tctgaaggaa tttatgcttt
     2641 ctcaagatgt tgaacatgag cgtctctttg acctcattca gaaaatgttg gagtatgatc
     2701 cagccaaaag aattactctc agagaagcct taaagcatcc tttctttgac cttctgaaga
     2761 aaagtatata cccaactttc ttgtacaaag tggttgatat ccagcacagt ggcggccgct
     2821 cgagtctaga gggcccgcgg ttcgaaacca gcggaggcgg cggaagcggg ggaggaggca
     2881 gcggcggagg cggcagcggg ggcggcggaa gcgctaccgg taagcctatc cctaaccctc
     2941 tcctcggtct cgattctacg cgtaccggta tggcttctaa ctttactcag ttcgttctcg
     3001 tcgacaatgg cggaactggc gacgtgactg tcgccccaag caacttcgct aacggggtcg
     3061 ctgaatggat cagctctaac tcgcgatcac aggcttacaa agtaacctgt agcgttcgtc
     3121 agagctctgc gcagaatcgc aaatacacca tcaaagtcga ggtgcctaaa gtggcaaccc
     3181 agactgttgg tggtgaagag cttcctgtag ccggatggag atcttactta aatatggaac
     3241 taaccattcc aattttcgcc acgaattccg actgcgagct tattgttaag gcaatgcaag
     3301 gtctcctaaa agatggaaac ccgattccct cggccatcgc ggccaactcc ggcatctacg
     3361 gaggtggagg tggatctcgt acgggttagt aatgagttta aacccgctga tcagcctcga
     3421 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     3481 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     3541 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     3601 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa
     3661 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg
     3721 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     3781 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     3841 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     3901 ttgattaggg tgatggttca cgtacctaga agttcctatt ccgaagttcc tattctctag
     3961 aaagtatagg aacttccttg gccaaaaagc ctgaactcac cgcgacgtct gtcgagaagt
     4021 ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag ggcgaagaat
     4081 ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta aatagctgcg
     4141 ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc gcgctcccga
     4201 ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc atctcccgcc
     4261 gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct gttctgcagc
     4321 cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg agcgggttcg
     4381 gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc atatgcgcga
     4441 ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg
     4501 tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc
     4561 tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc ataacagcgg
     4621 tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc aacatcttct
     4681 tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag cggaggcatc
     4741 cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt cttgaccaac
     4801 tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg
     4861 acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg
     4921 cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac cgacgcccca
     4981 gcactcgtcc gagggcaaag gaatagcacg tactacgaga tttcgattcc accgccgcct
     5041 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     5101 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     5161 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     5221 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     5281 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     5341 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     5401 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5461 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5521 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     5581 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     5641 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     5701 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     5761 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     5821 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     5881 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     5941 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     6001 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     6061 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     6121 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     6181 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     6241 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     6301 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     6361 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     6421 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     6481 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     6541 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     6601 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     6661 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     6721 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     6781 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     6841 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     6901 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     6961 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     7021 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     7081 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     7141 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     7201 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     7261 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     7321 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     7381 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     7441 cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc tatggtgcac
     7501 tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt
     7561 gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac
     7621 cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg
     7681 ggccagatat acgcgttgac attgattatt gactaggctt ttgcaaaaag ctttgcaaag
     7741 atggataaag ttttaaacag agaggaatct ttgcagctaa tggaccttct aggtcttgaa
     7801 aggagtggga att
//
