LOCUS       sequence-376767-        8455 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 8455)
  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 8455)
  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: GLE1
FEATURES             Location/Qualifiers
     source          1..8455
                     /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(3412..3436)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3561..3602
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(4057..4074)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4063..4287
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4420..4439)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4571..4618
                     /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             4626..5648
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4626..5648
                     /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    5778..5899
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5815..5834)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5869..5888
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5948..5964)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5948..5964)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5961..5983)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5972..5988
                     /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(5996..6002,6003..6020,6021..6026))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6041..6062
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6179..6196)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6350..6938)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6430..6449)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7109..7900,7901..7969))
                     /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     7732..7751
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7970..8074)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8149..8168)
                     /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 gttggcatgc
     1321 cgtctgaggg tcgctgctgg gagaccttga aggccctacg cagttccgac aaaggtcgcc
     1381 tttgctacta ccgcgactgg ctgctgcggc gcgaggatgt tttagaagaa tgtatgtctc
     1441 ttcccaagct atcttcttat tctggatggg tggtagagca cgtcctaccc catatgcagg
     1501 agaaccaacc tctgtctgag acttcgccat cctctacgtc agcttcagcc ctagatcaac
     1561 cctcatttgt tcccaaatct cctgacgcaa gctctgcctt ttccccagcc tcccctgcaa
     1621 caccaaatgg aaccaagggc aaagatgagt cccagcacac agaatctatg gtacttcagt
     1681 cctcacgggg gatcaaagtg gaagactgcg tccgaatgta cgaactggta cacagaatga
     1741 aaggaacaga gggcctgagg ctatggcagg aggagcagga gaggaaggtg caagccctct
     1801 cggagatggc atctgaacaa ctgaagcggt ttgatgaatg gaaggaactg aagcagcata
     1861 aagaattcca ggacttgcgg gaagtaatgg agaagagctc cagagaagcc ttgggacacc
     1921 aagagaagct aaaagctgag caccgtcaca gagcaaagat tctcaacctg aagctgcggg
     1981 aagcagagca gcagcgcgtg aagcaagcag aacaggagcg gcttcggaag gaagaaggcc
     2041 agatccgcct gcgggccctc tatgctctgc aggaggagat gctgcagctc agccagcagc
     2101 tggatgcctc tgagcagcac aaagccctgc ttaaggtcga cctggctgcc ttccagaccc
     2161 gaggcaacca gctgtgcagc ctcatctcag ggatcatccg ggcctcttca gagagcagct
     2221 atcccacagc agagagtcaa gctgaggctg agcgagctct gcgggaaatg cgggacctcc
     2281 tgatgaactt ggggcaggag atcaccagag cctgcgaaga caagaggagg caggatgaag
     2341 aagaggccca ggtaaagctg caagaggcac agatgcagca gggaccagag gcccacaaag
     2401 agcccccagc tcccagccag ggcccaggag ggaaacagaa tgaagacctc caggtgaagg
     2461 tacaagacat tacaatgcag tggtaccagc agctgcagga tgcttccatg cagtgtgtgt
     2521 tgacctttga gggcctgacc aacagcaagg acagtcaggc caaaaagata aagatggacc
     2581 tccagaaggc tgctaccatc ccagtgagcc aaatctctac cattgcaggc tcaaaactga
     2641 aggagatctt tgacaagatc cacagcctgc tctctggaaa acctgttcaa tctggtgggc
     2701 gctctgtgtc tgtcacactt aacccacagg ggctggactt tgttcaatac aaactggcag
     2761 agaaatttgt gaaacaaggc gaggaggaag tggcctctca ccatgaagca gcattcccca
     2821 ttgcagttgt ggcatccggg atctgggagc tccaccccag agtgggggac ctcattcttg
     2881 ctcatctaca taagaagtgt ccttactctg ttcctttcta tcccactttc aaggagggaa
     2941 tggctttgga agactatcag aggatgcttg gttaccaagt aaaggattcc aaagtggagc
     3001 agcaagacaa ctttctaaaa cgcatgtcag ggatgatccg tctctacgct gctatcatcc
     3061 agctccggtg gccatatgga aaccaacagg agattcaccc tcatggctta aatcatggat
     3121 ggcgctggtt ggcacagatc ttaaacatgg agcccttgtc agatgtgaca gccaccctcc
     3181 tctttgactt cctggaggtg tgtgggaatg ccctcatgaa gcaataccag gttcagttct
     3241 ggaagatgct aattctcatc aaagaggact actttcccag aattgaagct atcacaagct
     3301 caggacagat gggctccttc atacgcctca agcagttctt ggagaaatgt ttgcaacaca
     3361 aggacattcc tgtccccaag ggctttctga cttcctcctt ctggcgctcc tgcccaactt
     3421 tcttgtacaa agtggttgat atccagcaca gtggcggccg ctcgagtcta gagggcccgc
     3481 ggttcgaaac cagcggaggc ggcggaagcg ggggaggagg cagcggcgga ggcggcagcg
     3541 ggggcggcgg aagcgctacc ggtaagccta tccctaaccc tctcctcggt ctcgattcta
     3601 cgcgtaccgg tatggcttct aactttactc agttcgttct cgtcgacaat ggcggaactg
     3661 gcgacgtgac tgtcgcccca agcaacttcg ctaacggggt cgctgaatgg atcagctcta
     3721 actcgcgatc acaggcttac aaagtaacct gtagcgttcg tcagagctct gcgcagaatc
     3781 gcaaatacac catcaaagtc gaggtgccta aagtggcaac ccagactgtt ggtggtgaag
     3841 agcttcctgt agccggatgg agatcttact taaatatgga actaaccatt ccaattttcg
     3901 ccacgaattc cgactgcgag cttattgtta aggcaatgca aggtctccta aaagatggaa
     3961 acccgattcc ctcggccatc gcggccaact ccggcatcta cggaggtgga ggtggatctc
     4021 gtacgggtta gtaatgagtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
     4081 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     4141 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     4201 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     4261 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta
     4321 gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     4381 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     4441 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     4501 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     4561 cacgtaccta gaagttccta ttccgaagtt cctattctct agaaagtata ggaacttcct
     4621 tggccaaaaa gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg
     4681 acagcgtctc cgacctgatg cagctctcgg agggcgaaga atctcgtgct ttcagcttcg
     4741 atgtaggagg gcgtggatat gtcctgcggg taaatagctg cgccgatggt ttctacaaag
     4801 atcgttatgt ttatcggcac tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca
     4861 ttggggaatt cagcgagagc ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt
     4921 tgcaagacct gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg gaggccatgg
     4981 atgcgatcgc tgcggccgat cttagccaga cgagcgggtt cggcccattc ggaccgcaag
     5041 gaatcggtca atacactaca tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt
     5101 atcactggca aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg
     5161 agctgatgct ttgggccgag gactgccccg aagtccggca cctcgtgcac gcggatttcg
     5221 gctccaacaa tgtcctgacg gacaatggcc gcataacagc ggtcattgac tggagcgagg
     5281 cgatgttcgg ggattcccaa tacgaggtcg ccaacatctt cttctggagg ccgtggttgg
     5341 cttgtatgga gcagcagacg cgctacttcg agcggaggca tccggagctt gcaggatcgc
     5401 cgcggctccg ggcgtatatg ctccgcattg gtcttgacca actctatcag agcttggttg
     5461 acggcaattt cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg
     5521 gagccgggac tgtcgggcgt acacaaatcg cccgcagaag cgcggccgtc tggaccgatg
     5581 gctgtgtaga agtactcgcc gatagtggaa accgacgccc cagcactcgt ccgagggcaa
     5641 aggaatagca cgtactacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     5701 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     5761 agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     5821 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     5881 aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt
     5941 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     6001 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     6061 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     6121 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     6181 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     6241 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     6301 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     6361 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     6421 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     6481 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     6541 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     6601 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     6661 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     6721 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     6781 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     6841 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     6901 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     6961 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     7021 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     7081 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     7141 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     7201 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     7261 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     7321 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     7381 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     7441 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     7501 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     7561 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     7621 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     7681 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     7741 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     7801 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     7861 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     7921 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     7981 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     8041 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     8101 acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc
     8161 tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta
     8221 gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa
     8281 tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg
     8341 acattgatta ttgactaggc ttttgcaaaa agctttgcaa agatggataa agttttaaac
     8401 agagaggaat ctttgcagct aatggacctt ctaggtcttg aaaggagtgg gaatt
//
