LOCUS       sequence-376052-        7516 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 7516)
  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 7516)
  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: DCP2
FEATURES             Location/Qualifiers
     source          1..7516
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        67..1245
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          298..1236
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1193..1213
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     1262..1281
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1262..1280
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1359..1383
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2539..2563)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2688..2729
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3184..3201)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3190..3414
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3547..3566)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3698..3745
                     /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             3753..4775
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3753..4775
                     /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    4905..5026
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4942..4961)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4996..5015
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5075..5091)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5075..5091)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5088..5110)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5099..5115
                     /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(5123..5129,5130..5147,5148..5153))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5168..5189
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5306..5323)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5477..6065)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5557..5576)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6236..7027,7028..7096))
                     /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     6859..6878
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7097..7201)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7276..7295)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aagttttaaa cagagaggaa tctttgcagc taatggacct tctaggtctt gaaaggagtg
       61 ggaattggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag
      121 ttggggggag gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg
      181 gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata
      241 agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggta
      301 agtgccgtgt gtggttcccg cgggcctggc ctctttacgg gttatggccc ttgcgtgcct
      361 tgaattactt ccacctggct gcagtacgtg attcttgatc ccgagcttcg ggttggaagt
      421 gggtgggaga gttcgaggcc ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag
      481 gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc
      541 gctgctttcg ataagtctct agccatttaa aatttttgat gacctgctgc gacgcttttt
      601 ttctggcaag atagtcttgt aaatgcgggc caagatctgc acactggtat ttcggttttt
      661 ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca catgttcggc gaggcggggc
      721 ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc aagctggccg gcctgctctg
      781 gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg
      841 gcaccagttg cgtgagcgga aagatggccg cttcccggcc ctgctgcagg gagctcaaaa
      901 tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc
      961 tttccgtcct cagccgtcgc ttcatgtgac tccacggagt accgggcgcc gtccaggcac
     1021 ctcgattagt tctcgagctt ttggagtacg tcgtctttag gttgggggga ggggttttat
     1081 gcgatggagt ttccccacac tgagtgggtg gagactgaag ttaggccagc ttggcacttg
     1141 atgtaattct ccttggaatt tgcccttttt gagtttggat cttggttcat tctcaagcct
     1201 cagacagtgg ttcaaagttt ttttcttcca tttcaggtgt cgtgaggaat tagcttggta
     1261 ctaatacgac tcactatagg gagacccaag ctggctaggt aagcttggta ccgagctcgg
     1321 atccactagt ccagtgtggt ggaattctgc agatatcaac aagtttgtac aaaaaagttg
     1381 gcatggagac caaacgggtg gagattcccg gcagcgtcct ggacgatctc tgcagccgat
     1441 ttattttgca tattcccagc gaggaaagag acaatgcaat ccgagtgtgt tttcagattg
     1501 aacttgccca ttggttttac ttggatttct acatgcagaa cacaccagga ttacctcagt
     1561 gtgggataag agactttgct aaagctgtct tcagtcattg tccgtttttg ctgcctcaag
     1621 gtgaagatgt ggaaaaagtt ttggatgaat ggaaggaata taaaatggga gtaccaacat
     1681 atggtgcaat tattcttgat gagacacttg aaaatgtact actagttcag gggtacctag
     1741 caaaatcagg ctggggattt ccaaaaggaa aagtaaataa agaagaagct cctcatgatt
     1801 gtgctgctag agaggtcttt gaagaaactg gttttgatat caaagactat atttgtaagg
     1861 atgattacat tgaacttcga atcaatgacc agcttgctcg tttgtacatc attccaggaa
     1921 ttccaaaaga cacaaaattt aacccaaaaa ctagaagaga aattcggaac attgagtggt
     1981 tctctattga gaaattgcct tgtcatagaa atgatatgac ccccaaatcc aaacttggtt
     2041 tggcacctaa caaatttttt atggccattc cctttatcag accattaagg gactggcttt
     2101 ctcgaagatt tggcgattcc tcagacagtg acaatggatt ttcctcaact ggtagcacgc
     2161 cggctaaacc cactgtggaa aaattgagtc gaaccaaatt ccgccacagt cagcagttat
     2221 ttcctgacgg ttctcctggt gaccagtggg taaagcacag gcaaccactg cagcaaaagc
     2281 catataataa tcattctgaa atgtctgacc ttttaaaagg aaagaagtgt gaaaagaaac
     2341 ttcatccacg gaaacttcag gataattttg aaacagatgc tgtatatgac ttgcctagct
     2401 ccagtgaaga ccagttgcta gaacatgctg agggacagcc cgtggcatgt aatggacatt
     2461 gcaagttccc cttttcatcc agagcctttt tgagtttcaa gtttgaccat aatgctataa
     2521 tgaaaatctt ggacctttgc ccaactttct tgtacaaagt ggttgatatc cagcacagtg
     2581 gcggccgctc gagtctagag ggcccgcggt tcgaaaccag cggaggcggc ggaagcgggg
     2641 gaggaggcag cggcggaggc ggcagcgggg gcggcggaag cgctaccggt aagcctatcc
     2701 ctaaccctct cctcggtctc gattctacgc gtaccggtat ggcttctaac tttactcagt
     2761 tcgttctcgt cgacaatggc ggaactggcg acgtgactgt cgccccaagc aacttcgcta
     2821 acggggtcgc tgaatggatc agctctaact cgcgatcaca ggcttacaaa gtaacctgta
     2881 gcgttcgtca gagctctgcg cagaatcgca aatacaccat caaagtcgag gtgcctaaag
     2941 tggcaaccca gactgttggt ggtgaagagc ttcctgtagc cggatggaga tcttacttaa
     3001 atatggaact aaccattcca attttcgcca cgaattccga ctgcgagctt attgttaagg
     3061 caatgcaagg tctcctaaaa gatggaaacc cgattccctc ggccatcgcg gccaactccg
     3121 gcatctacgg aggtggaggt ggatctcgta cgggttagta atgagtttaa acccgctgat
     3181 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     3241 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     3301 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     3361 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
     3421 aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat
     3481 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     3541 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     3601 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     3661 ccaaaaaact tgattagggt gatggttcac gtacctagaa gttcctattc cgaagttcct
     3721 attctctaga aagtatagga acttccttgg ccaaaaagcc tgaactcacc gcgacgtctg
     3781 tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag ctctcggagg
     3841 gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc ctgcgggtaa
     3901 atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt gcatcggccg
     3961 cgctcccgat tccggaagtg cttgacattg gggaattcag cgagagcctg acctattgca
     4021 tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg
     4081 ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc ggccgatctt agccagacga
     4141 gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg cgtgatttca
     4201 tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac gacaccgtca
     4261 gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac tgccccgaag
     4321 tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac aatggccgca
     4381 taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac gaggtcgcca
     4441 acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc tacttcgagc
     4501 ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc cgcattggtc
     4561 ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct tgggcgcagg
     4621 gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca caaatcgccc
     4681 gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat agtggaaacc
     4741 gacgccccag cactcgtccg agggcaaagg aatagcacgt actacgagat ttcgattcca
     4801 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     4861 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     4921 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4981 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     5041 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     5101 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     5161 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     5221 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5281 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     5341 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     5401 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     5461 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     5521 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     5581 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     5641 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     5701 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5761 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5821 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5881 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     5941 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     6001 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     6061 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     6121 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     6181 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     6241 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     6301 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     6361 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     6421 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     6481 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     6541 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6601 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     6661 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6721 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6781 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6841 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6901 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6961 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     7021 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     7081 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     7141 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     7201 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct
     7261 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc
     7321 tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca
     7381 aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg
     7441 cgatgtacgg gccagatata cgcgttgaca ttgattattg actaggcttt tgcaaaaagc
     7501 tttgcaaaga tggata
//
