LOCUS       sequence-376717-        7990 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 7990)
  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 7990)
  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: CNOT2
FEATURES             Location/Qualifiers
     source          1..7990
                     /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(2947..2971)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3096..3137
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3592..3609)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3598..3822
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3955..3974)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4106..4153
                     /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             4161..5183
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4161..5183
                     /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    5313..5434
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5350..5369)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5404..5423
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5483..5499)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5483..5499)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5496..5518)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5507..5523
                     /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(5531..5537,5538..5555,5556..5561))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5576..5597
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5714..5731)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5885..6473)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5965..5984)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6644..7435,7436..7504))
                     /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     7267..7286
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7505..7609)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7684..7703)
                     /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 gcaggctcca
     1321 ccatggtgag gactgatgga catacattat ctgagaaaag aaactaccag gtgacaaaca
     1381 gcatgtttgg tgcttcaaga aagaagtttg tagagggggt cgacagtgac taccatgacg
     1441 aaaacatgta ctacagccag tcttctatgt ttccacatcg gtcagaaaaa gatatgctgg
     1501 catcaccatc tacatcaggt cagctgtctc agtttggggc aagtttatac gggcaacaaa
     1561 gtgcactagg ccttccaatg agggggatga gcaacaatac ccctcagtta aatcgcagct
     1621 tatcacaagg cactcagtta ccgagccacg tcacgccaac aacaggggta ccaacaatgt
     1681 cacttcacac gcctccatct ccaagcaggg gtattttgcc tatgaatcct aggaatatga
     1741 tgaaccactc ccaggttggt cagggcattg gaattcctag caggacaaat agcatgagca
     1801 gttcagggtt aggtagcccc aacagaagct cgccaagcat aatatgtatg ccaaagcagc
     1861 agccttctcg acagcctttt actgtgaaca gtatgtctgg atttggaatg aacaggaatc
     1921 aggcatttgg aatgaataac tccttatcaa gtaacatttt taatggaaca gacggaagtg
     1981 aaaatgtgac aggattggac ctttcagatt tcccagcatt agcagaccga aacaggaggg
     2041 aaggaagtgg taacccaact ccattaataa accccttggc tggaagagct ccttatgttg
     2101 gaatggtaac aaaaccagca aatgaacaat cccaggactt ctcaatacac aatgaagatt
     2161 ttccagcatt accaggctcc agctataaag atccaacatc aagtaatgat gacagtaaat
     2221 ctaatttgaa tacatctggc aagacaactt caagtacaga tggacccaaa ttccctggag
     2281 ataaaagttc aacaacacaa aataataacc agcagaaaaa agggatccag gtgttacctg
     2341 atggtcgggt tactaacatt cctcaaggga tggtgacgga ccaatttgga atgattggcc
     2401 tgttaacatt tatcagggca gcagagacag acccaggaat ggtacatctt gcattaggaa
     2461 gtgacttaac aacattaggc ctcaatctga actctcctga aaatctctac cccaaatttg
     2521 cgtcaccctg ggcatcttca ccttgtcgac ctcaagacat agacttccat gttccatctg
     2581 agtacttaac gaacattcac attagggata agctggctgc aataaaactt ggccgatatg
     2641 gtgaagacct tctcttctat ctctattaca tgaatggagg agacgtatta caacttttag
     2701 ctgcagtgga gctttttaac cgtgattgga gataccacaa agaagaacga gtatggatta
     2761 ccagggcacc aggcatggag ccaacaatga aaaccaatac ctatgagagg ggaacatatt
     2821 acttctttga ctgtcttaac tggaggaaag tagctaagga gttccatctg gaatatgaca
     2881 aattagaaga acggcctcac ctgccatcca ccttcaacta caaccctgct cagcaagcct
     2941 tcttggaccc agctttcttg tacaaagtgg ttgatatcca gcacagtggc ggccgctcga
     3001 gtctagaggg cccgcggttc gaaaccagcg gaggcggcgg aagcggggga ggaggcagcg
     3061 gcggaggcgg cagcgggggc ggcggaagcg ctaccggtaa gcctatccct aaccctctcc
     3121 tcggtctcga ttctacgcgt accggtatgg cttctaactt tactcagttc gttctcgtcg
     3181 acaatggcgg aactggcgac gtgactgtcg ccccaagcaa cttcgctaac ggggtcgctg
     3241 aatggatcag ctctaactcg cgatcacagg cttacaaagt aacctgtagc gttcgtcaga
     3301 gctctgcgca gaatcgcaaa tacaccatca aagtcgaggt gcctaaagtg gcaacccaga
     3361 ctgttggtgg tgaagagctt cctgtagccg gatggagatc ttacttaaat atggaactaa
     3421 ccattccaat tttcgccacg aattccgact gcgagcttat tgttaaggca atgcaaggtc
     3481 tcctaaaaga tggaaacccg attccctcgg ccatcgcggc caactccggc atctacggag
     3541 gtggaggtgg atctcgtacg ggttagtaat gagtttaaac ccgctgatca gcctcgactg
     3601 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     3661 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     3721 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     3781 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     3841 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
     3901 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     3961 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     4021 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     4081 attagggtga tggttcacgt acctagaagt tcctattccg aagttcctat tctctagaaa
     4141 gtataggaac ttccttggcc aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc
     4201 tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc gaagaatctc
     4261 gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg
     4321 atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg ctcccgattc
     4381 cggaagtgct tgacattggg gaattcagcg agagcctgac ctattgcatc tcccgccgtg
     4441 cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg
     4501 tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc gggttcggcc
     4561 cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata tgcgcgattg
     4621 ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt gcgtccgtcg
     4681 cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc cggcacctcg
     4741 tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata acagcggtca
     4801 ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac atcttcttct
     4861 ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg aggcatccgg
     4921 agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt gaccaactct
     4981 atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg
     5041 caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg
     5101 ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga cgccccagca
     5161 ctcgtccgag ggcaaaggaa tagcacgtac tacgagattt cgattccacc gccgccttct
     5221 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     5281 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     5341 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     5401 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     5461 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     5521 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     5581 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     5641 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     5701 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     5761 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     5821 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     5881 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     5941 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     6001 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     6061 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     6121 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     6181 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     6241 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     6301 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     6361 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     6421 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     6481 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     6541 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     6601 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     6661 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     6721 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     6781 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     6841 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     6901 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     6961 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     7021 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     7081 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     7141 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     7201 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     7261 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     7321 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     7381 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     7441 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     7501 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     7561 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     7621 gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct
     7681 cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
     7741 ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga
     7801 caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
     7861 cagatatacg cgttgacatt gattattgac taggcttttg caaaaagctt tgcaaagatg
     7921 gataaagttt taaacagaga ggaatctttg cagctaatgg accttctagg tcttgaaagg
     7981 agtgggaatt
//
