LOCUS       sequence-376027-        6988 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 6988)
  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 6988)
  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: BTG4
FEATURES             Location/Qualifiers
     source          1..6988
                     /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(1945..1969)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2094..2135
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2590..2607)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2596..2820
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2953..2972)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3104..3151
                     /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             3159..4181
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3159..4181
                     /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    4311..4432
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4348..4367)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4402..4421
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4481..4497)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4481..4497)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4494..4516)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4505..4521
                     /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(4529..4535,4536..4553,4554..4559))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4574..4595
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4712..4729)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4883..5471)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4963..4982)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5642..6433,6434..6502))
                     /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     6265..6284
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6503..6607)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6682..6701)
                     /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 ccatgagaga tgaaattgca acaacagttt tctttgtcac aagattggtg aaaaaacatg
     1381 ataaactaag taaacagcaa atagaagact ttgcagaaaa gctgatgacg atcttgtttg
     1441 aaacatacag aagtcactgg cactctgatt gcccttctaa agggcaagcc ttcaggtgca
     1501 tcaggataaa caacaatcag aataaagatc ccattctaga aagggcatgt gtggaaagta
     1561 atgtagattt ttctcacctg ggacttccga aggagatgac catatgggta gatccctttg
     1621 aagtatgctg taggtatggt gagaaaaacc atccatttac agttgcttct tttaaaggca
     1681 gatgggagga atgggaacta tatcaacaaa tcagttatgc agttagtaga gcctcatcag
     1741 acgtttcctc tggcacttcc tgcgatgaag aaagttgtag caaggaacct cgtgtcattc
     1801 ctaaagtcag caatccgaag agtatttatc aggtcaagag tgttccagtt cttttctata
     1861 ctttttttct atctaattct aaaaagaatg cactcattat gaaaacaaag aagcaaaaaa
     1921 acatggaaag aacaaaactg ttggacccag ctttcttgta caaagtggtt gatatccagc
     1981 acagtggcgg ccgctcgagt ctagagggcc cgcggttcga aaccagcgga ggcggcggaa
     2041 gcgggggagg aggcagcggc ggaggcggca gcgggggcgg cggaagcgct accggtaagc
     2101 ctatccctaa ccctctcctc ggtctcgatt ctacgcgtac cggtatggct tctaacttta
     2161 ctcagttcgt tctcgtcgac aatggcggaa ctggcgacgt gactgtcgcc ccaagcaact
     2221 tcgctaacgg ggtcgctgaa tggatcagct ctaactcgcg atcacaggct tacaaagtaa
     2281 cctgtagcgt tcgtcagagc tctgcgcaga atcgcaaata caccatcaaa gtcgaggtgc
     2341 ctaaagtggc aacccagact gttggtggtg aagagcttcc tgtagccgga tggagatctt
     2401 acttaaatat ggaactaacc attccaattt tcgccacgaa ttccgactgc gagcttattg
     2461 ttaaggcaat gcaaggtctc ctaaaagatg gaaacccgat tccctcggcc atcgcggcca
     2521 actccggcat ctacggaggt ggaggtggat ctcgtacggg ttagtaatga gtttaaaccc
     2581 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     2641 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     2701 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     2761 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
     2821 cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg
     2881 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     2941 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     3001 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     3061 tcgaccccaa aaaacttgat tagggtgatg gttcacgtac ctagaagttc ctattccgaa
     3121 gttcctattc tctagaaagt ataggaactt ccttggccaa aaagcctgaa ctcaccgcga
     3181 cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg atgcagctct
     3241 cggagggcga agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc
     3301 gggtaaatag ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat
     3361 cggccgcgct cccgattccg gaagtgcttg acattgggga attcagcgag agcctgacct
     3421 attgcatctc ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa accgaactgc
     3481 ccgctgttct gcagccggtc gcggaggcca tggatgcgat cgctgcggcc gatcttagcc
     3541 agacgagcgg gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg
     3601 atttcatatg cgcgattgct gatccccatg tgtatcactg gcaaactgtg atggacgaca
     3661 ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc
     3721 ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa caatgtcctg acggacaatg
     3781 gccgcataac agcggtcatt gactggagcg aggcgatgtt cggggattcc caatacgagg
     3841 tcgccaacat cttcttctgg aggccgtggt tggcttgtat ggagcagcag acgcgctact
     3901 tcgagcggag gcatccggag cttgcaggat cgccgcggct ccgggcgtat atgctccgca
     3961 ttggtcttga ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg
     4021 cgcagggtcg atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa
     4081 tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt agaagtactc gccgatagtg
     4141 gaaaccgacg ccccagcact cgtccgaggg caaaggaata gcacgtacta cgagatttcg
     4201 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     4261 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta
     4321 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     4381 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     4441 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     4501 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     4561 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     4621 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     4681 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     4741 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4801 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     4861 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     4921 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     4981 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     5041 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     5101 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     5161 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5221 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     5281 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     5341 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     5401 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     5461 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     5521 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     5581 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     5641 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     5701 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     5761 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     5821 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     5881 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     5941 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     6001 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     6061 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     6121 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     6181 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     6241 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     6301 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     6361 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     6421 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     6481 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     6541 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     6601 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga
     6661 tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct
     6721 gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac
     6781 aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct
     6841 gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta ggcttttgca
     6901 aaaagctttg caaagatgga taaagtttta aacagagagg aatctttgca gctaatggac
     6961 cttctaggtc ttgaaaggag tgggaatt
//
