LOCUS       sequence-376744-        7363 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 7363)
  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 7363)
  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: RSRC1
FEATURES             Location/Qualifiers
     source          1..7363
                     /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(2320..2344)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2469..2510
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2965..2982)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2971..3195
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3328..3347)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3479..3526
                     /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             3534..4556
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3534..4556
                     /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    4686..4807
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4723..4742)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4777..4796
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4856..4872)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4856..4872)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4869..4891)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4880..4896
                     /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(4904..4910,4911..4928,4929..4934))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4949..4970
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5087..5104)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5258..5846)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5338..5357)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6017..6808,6809..6877))
                     /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     6640..6659
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6878..6982)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7057..7076)
                     /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 gacgtcggtc atcagatact gaagaagaaa gcagaagcaa gagaaaaaag aaacaccgta
     1381 gacggtcctc ctcgagcagt tcttcagata gtagaacata cagccgaaag aaaggaggaa
     1441 ggaaatcaag atcaaagtca agatcttggt ccagagatct tcagcctcgt tcacattctt
     1501 atgatagaag acgcaggcat cgatcaagca gtagctcttc ttatggctcc agaaggaaac
     1561 gaagtcgaag tcgttcaagg ggtcgaggga aatcctatag agttcagagg tctaggtcaa
     1621 aaagcagaac aagaaggtcc aggtcaagac ctcgtctccg ttctcatagt cgtagcagtg
     1681 aaaggtccag tcacagaaga acgcgtagtc ggtctcggga tagagaacga cgtaagggca
     1741 gagataaaga gaaaagagaa aaggagaagg ataaagggaa ggacaaggaa ttacataaca
     1801 tcaaacgtgg ggaatctgga aacatcaaag ctggattaga acatctgcca ccagctgaac
     1861 aggccaaagc cagactacag ctggttcttg aagctgctgc aaaagctgat gaagcattga
     1921 aagccaaaga aagaaatgag gaagaagcaa agagaagaaa ggaggaagac caagccaccc
     1981 tggtagaaca agtaaaaaga gtaaaagaaa ttgaagctat tgaaagtgat tcttttgttc
     2041 agcagacatt cagatcaagt aaagaagtca aaaagtcagt ggaacctagt gaagtgaaac
     2101 aagcaacttc aacatcagga ccagcatcag cagttgctga tccacccagt actgaaaaag
     2161 aaatagatcc taccagcatc cctactgcta tcaagtacca agatgacaat tccctggccc
     2221 atccaaattt atttatcgag aaagctgatg ctgaggaaaa atggttcaag agattaattg
     2281 ctctccgaca agaaagacta atgggcagtc ctgtggccta cccaactttc ttgtacaaag
     2341 tggttgatat ccagcacagt ggcggccgct cgagtctaga gggcccgcgg ttcgaaacca
     2401 gcggaggcgg cggaagcggg ggaggaggca gcggcggagg cggcagcggg ggcggcggaa
     2461 gcgctaccgg taagcctatc cctaaccctc tcctcggtct cgattctacg cgtaccggta
     2521 tggcttctaa ctttactcag ttcgttctcg tcgacaatgg cggaactggc gacgtgactg
     2581 tcgccccaag caacttcgct aacggggtcg ctgaatggat cagctctaac tcgcgatcac
     2641 aggcttacaa agtaacctgt agcgttcgtc agagctctgc gcagaatcgc aaatacacca
     2701 tcaaagtcga ggtgcctaaa gtggcaaccc agactgttgg tggtgaagag cttcctgtag
     2761 ccggatggag atcttactta aatatggaac taaccattcc aattttcgcc acgaattccg
     2821 actgcgagct tattgttaag gcaatgcaag gtctcctaaa agatggaaac ccgattccct
     2881 cggccatcgc ggccaactcc ggcatctacg gaggtggagg tggatctcgt acgggttagt
     2941 aatgagttta aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     3001 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     3061 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     3121 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
     3181 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc
     3241 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     3301 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     3361 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     3421 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtacctaga
     3481 agttcctatt ccgaagttcc tattctctag aaagtatagg aacttccttg gccaaaaagc
     3541 ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg
     3601 acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc
     3661 gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt
     3721 atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattca
     3781 gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc
     3841 ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat gcgatcgctg
     3901 cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat
     3961 acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa
     4021 ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt
     4081 gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg
     4141 tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg
     4201 attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc
     4261 agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg cggctccggg
     4321 cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg
     4381 atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg
     4441 tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag
     4501 tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaatagcacg
     4561 tactacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     4621 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     4681 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     4741 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     4801 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
     4861 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     4921 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     4981 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     5041 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     5101 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     5161 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     5221 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     5281 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     5341 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     5401 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     5461 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     5521 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     5581 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     5641 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     5701 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     5761 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     5821 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     5881 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     5941 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     6001 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     6061 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     6121 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     6181 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     6241 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     6301 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     6361 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     6421 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     6481 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6541 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     6601 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     6661 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     6721 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     6781 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     6841 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     6901 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     6961 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat
     7021 cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata
     7081 gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa
     7141 aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt
     7201 taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac attgattatt
     7261 gactaggctt ttgcaaaaag ctttgcaaag atggataaag ttttaaacag agaggaatct
     7321 ttgcagctaa tggaccttct aggtcttgaa aggagtggga att
//
