LOCUS       sequence-376055-        8224 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 8224)
  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 8224)
  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: SR140
FEATURES             Location/Qualifiers
     source          1..8224
                     /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(3181..3205)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3330..3371
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3826..3843)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3832..4056
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4189..4208)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4340..4387
                     /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             4395..5417
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4395..5417
                     /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    5547..5668
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5584..5603)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5638..5657
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5717..5733)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5717..5733)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5730..5752)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5741..5757
                     /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(5765..5771,5772..5789,5790..5795))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5810..5831
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5948..5965)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6119..6707)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6199..6218)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6878..7669,7670..7738))
                     /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     7501..7520
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7739..7843)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7918..7937)
                     /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 gttggcacca
     1321 tgctcctatg ttaccgccac ctaaaaacaa agaggatttt gagaaggaat ttgctcgccc
     1381 tgatacatcg aatgattgag tttgttgtac gtgaagggcc aatgtttgaa gctatgatta
     1441 tgaacagaga aatcaacaat cctatgttca ggttcttatt tgaaaaccag acaccagccc
     1501 atgtttacta taggtggaag ctttattcta ttctgcaggg agattctcca actaaatggc
     1561 ggacggaaga ttttcgtatg ttcaaaaatg gatctttttg gaggccacca ccattaaatc
     1621 cgtacttgca tggaatgtca gaagagcaag aaacagaagc ttttgtagag gaacctagta
     1681 aaaagggagc acttaaggaa gaacagaggg ataaattgga agaaatcttg cggggattaa
     1741 ctccaaggaa aaatgatatt ggagatgcaa tggttttctg tcttaataat gctgaagctg
     1801 ctgaagaaat agtggattgc attactgagt cgttgtccat cttaaagaca ccccttccta
     1861 aaaagattgc cagattatat ttggtttctg atgttttgta caactcttca gccaaagttg
     1921 ctaatgcttc atattataga aaattttttg aaacaaagtt atgtcagata ttttcagacc
     1981 tcaatgccac ctatcgtaca attcaaggcc atttacaatc tgaaaacttt aagcaacggg
     2041 taatgacttg cttcagagca tgggaagatt gggcaattta tccagaacca tttttgatca
     2101 aactacaaaa tattttctta ggacttgtaa atattattga agaaaaggaa acagaggatg
     2161 ttccagatga ccttgatggt gcccccatcg aggaagagct tgatggtgca cctctggaag
     2221 atgtagatgg aattcctatt gatgctactc ccatcgatga tcttgatgga gtccctataa
     2281 aaagtcttga tgatgatctt gatggagtgc ctttggatgc aactgaagac tcaaaaaaga
     2341 atgagcctat atttaaagtt gccccatcaa aatgggaagc tgtggatgaa tctgaattgg
     2401 aagcacaggc tgttacaact tctaaatggg aattatttga ccagcatgaa gaatcagaag
     2461 aagaagaaaa tcaaaatcaa gaagaagaaa gtgaagatga agaagatact caaagttcca
     2521 aatctgaaga acatcatttg tactctaatc caatcaaaga agaaatgact gagtctaagt
     2581 tctctaagta ctctgaaatg agtgaggaaa aacgagccaa acttcgtgaa attgagctca
     2641 aagttatgaa gtttcaggat gaattggaat ctgggaaaag acctaaaaaa ccaggccaga
     2701 gttttcagga gcaagtagaa cactacagag ataaacttct tcaacgagag aaagagaaag
     2761 agttagaaag agaacgagaa agagacaaga aagataaaga aaaattggaa tctcgctcca
     2821 aagacaagaa ggaaaaagat gagtgtactc cgacaaggaa ggaaaggaag aggcgacaca
     2881 gtacatcccc cagcccatct cgcagtagca gtggtagacg agtgaaatcc ccatcaccaa
     2941 aatcggagcg atcagagcgt tcagaaagat ctcataaaga gagctcacgg tccaggtcat
     3001 ctcacaaaga ttctcctaga gatgttagca aaaaagccaa aagatcacca tctggttcaa
     3061 ggacacctaa aaggtctagg cgatcacggt ctagatctcc taaaaaatca ggaaagaagt
     3121 ccagatccca gtccagatct ccacacaggt ctcataaaaa gtcaaagaaa aacaaacact
     3181 tgccaacttt cttgtacaaa gtggttgata tccagcacag tggcggccgc tcgagtctag
     3241 agggcccgcg gttcgaaacc agcggaggcg gcggaagcgg gggaggaggc agcggcggag
     3301 gcggcagcgg gggcggcgga agcgctaccg gtaagcctat ccctaaccct ctcctcggtc
     3361 tcgattctac gcgtaccggt atggcttcta actttactca gttcgttctc gtcgacaatg
     3421 gcggaactgg cgacgtgact gtcgccccaa gcaacttcgc taacggggtc gctgaatgga
     3481 tcagctctaa ctcgcgatca caggcttaca aagtaacctg tagcgttcgt cagagctctg
     3541 cgcagaatcg caaatacacc atcaaagtcg aggtgcctaa agtggcaacc cagactgttg
     3601 gtggtgaaga gcttcctgta gccggatgga gatcttactt aaatatggaa ctaaccattc
     3661 caattttcgc cacgaattcc gactgcgagc ttattgttaa ggcaatgcaa ggtctcctaa
     3721 aagatggaaa cccgattccc tcggccatcg cggccaactc cggcatctac ggaggtggag
     3781 gtggatctcg tacgggttag taatgagttt aaacccgctg atcagcctcg actgtgcctt
     3841 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     3901 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     3961 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     4021 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     4081 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     4141 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     4201 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     4261 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     4321 gtgatggttc acgtacctag aagttcctat tccgaagttc ctattctcta gaaagtatag
     4381 gaacttcctt ggccaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg
     4441 aaaagttcga cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt
     4501 tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt
     4561 tctacaaaga tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag
     4621 tgcttgacat tggggaattc agcgagagcc tgacctattg catctcccgc cgtgcacagg
     4681 gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg
     4741 aggccatgga tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg
     4801 gaccgcaagg aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc
     4861 cccatgtgta tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg
     4921 ctctcgatga gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg
     4981 cggatttcgg ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact
     5041 ggagcgaggc gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc
     5101 cgtggttggc ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg
     5161 caggatcgcc gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga
     5221 gcttggttga cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg
     5281 tccgatccgg agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct
     5341 ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc
     5401 cgagggcaaa ggaatagcac gtactacgag atttcgattc caccgccgcc ttctatgaaa
     5461 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     5521 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     5581 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     5641 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     5701 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     5761 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     5821 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     5881 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     5941 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     6001 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     6061 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     6121 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     6181 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     6241 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     6301 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     6361 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     6421 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     6481 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     6541 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     6601 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     6661 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     6721 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     6781 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     6841 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     6901 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     6961 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     7021 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     7081 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     7141 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     7201 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     7261 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     7321 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     7381 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     7441 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     7501 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     7561 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     7621 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     7681 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     7741 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     7801 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     7861 tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac
     7921 aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt
     7981 cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg
     8041 catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata
     8101 tacgcgttga cattgattat tgactaggct tttgcaaaaa gctttgcaaa gatggataaa
     8161 gttttaaaca gagaggaatc tttgcagcta atggaccttc taggtcttga aaggagtggg
     8221 aatt
//
