LOCUS       sequence-377218-        7558 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 7558)
  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 7558)
  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: SF3B3-1
FEATURES             Location/Qualifiers
     source          1..7558
                     /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"
     CDS             complement(1417..1428)
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     protein_bind    complement(2515..2539)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2664..2705
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3160..3177)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3166..3390
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3523..3542)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3674..3721
                     /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             3729..4751
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3729..4751
                     /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    4881..5002
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4918..4937)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4972..4991
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5051..5067)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5051..5067)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5064..5086)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5075..5091
                     /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(5099..5105,5106..5123,5124..5129))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5144..5165
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5282..5299)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5453..6041)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5533..5552)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6212..7003,7004..7072))
                     /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     6835..6854
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7073..7177)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7252..7271)
                     /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 cagaggaaat ggtggaagca gcaggggagg atgagcggga gctggccgca gagatggcag
     1381 cagcattcct caatgaaaac ctccctgaat ccatctttgg agctcccaag gctggcaatg
     1441 ggcagtgggc ctctgtgatc cgagtgatga atcccattca agggaacaca ctggaccttg
     1501 tccagctgga acagaatgag gcagctttta gtgtggctgt gtgcaggttt tccaacattg
     1561 gtgaagactg gtatgtgctg gtgggtgtgg ccaaggacct gatactaaac ccccgatctg
     1621 tggcaggggg cttcgtctat acttacaagc ttgtgaacaa tggggaaaaa ctggagtttt
     1681 tgcacaagac tcctgtggaa gaggtccctg ctgctattgc cccattccag gggagggtgt
     1741 tgattggtgt ggggaagctg ttgcgtgtct atgacctggg aaagaagaag ttactccgaa
     1801 aatgtgagaa taagcatatt gccaattata tctctgggat ccagactatc ggacataggg
     1861 taattgtatc tgatgtccaa gaaagtttca tctgggttcg ctacaagcgt aatgaaaacc
     1921 agcttatcat ctttgctgat gatacctacc cccgatgggt cactacagcc agcctcctgg
     1981 actatgacac tgtggctggg gcagacaagt ttggcaacat atgtgtggtg aggctcccac
     2041 ctaacaccaa tgatgaagta gatgaggatc ctacaggaaa caaagccctg tgggaccgtg
     2101 gcttgctcaa tggggcctcc cagaaggcag aggtgatcat gaactaccat gtcggggaga
     2161 cggtgctgtc cttgcagaag accacgctga tccctggagg ctcagaatca cttgtctata
     2221 ccaccttgtc tggaggaatt ggcatccttg tgccattcac gtcccatgag gaccatgact
     2281 tcttccagca tgtggaaatg cacctgcggt ctgaacatcc ccctctctgt gggcgggacc
     2341 acctcagctt tcgctcctac tacttccctg tgaagaatgt gattgatgga gacctctgtg
     2401 agcagttcaa ttccatggaa cccaacaaac aaaagaacgt ctctgaagaa ctggaccgaa
     2461 ccccacccga agtgtccaag aaactcgagg atatccggac ccgctacgcc ttctgcccaa
     2521 ctttcttgta caaagtggtt gatatccagc acagtggcgg ccgctcgagt ctagagggcc
     2581 cgcggttcga aaccagcgga ggcggcggaa gcgggggagg aggcagcggc ggaggcggca
     2641 gcgggggcgg cggaagcgct accggtaagc ctatccctaa ccctctcctc ggtctcgatt
     2701 ctacgcgtac cggtatggct tctaacttta ctcagttcgt tctcgtcgac aatggcggaa
     2761 ctggcgacgt gactgtcgcc ccaagcaact tcgctaacgg ggtcgctgaa tggatcagct
     2821 ctaactcgcg atcacaggct tacaaagtaa cctgtagcgt tcgtcagagc tctgcgcaga
     2881 atcgcaaata caccatcaaa gtcgaggtgc ctaaagtggc aacccagact gttggtggtg
     2941 aagagcttcc tgtagccgga tggagatctt acttaaatat ggaactaacc attccaattt
     3001 tcgccacgaa ttccgactgc gagcttattg ttaaggcaat gcaaggtctc ctaaaagatg
     3061 gaaacccgat tccctcggcc atcgcggcca actccggcat ctacggaggt ggaggtggat
     3121 ctcgtacggg ttagtaatga gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt
     3181 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     3241 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     3301 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     3361 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
     3421 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     3481 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     3541 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     3601 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     3661 gttcacgtac ctagaagttc ctattccgaa gttcctattc tctagaaagt ataggaactt
     3721 ccttggccaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt
     3781 tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct
     3841 tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca
     3901 aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg
     3961 acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca
     4021 cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca
     4081 tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc
     4141 aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg
     4201 tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg
     4261 atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt
     4321 tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg
     4381 aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt
     4441 tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat
     4501 cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg
     4561 ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat
     4621 ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg
     4681 atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg
     4741 caaaggaata gcacgtacta cgagatttcg attccaccgc cgccttctat gaaaggttgg
     4801 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     4861 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     4921 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     4981 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     5041 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     5101 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     5161 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     5221 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     5281 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     5341 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     5401 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     5461 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     5521 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     5581 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     5641 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     5701 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     5761 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     5821 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     5881 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     5941 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     6001 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     6061 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     6121 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     6181 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     6241 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     6301 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     6361 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     6421 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     6481 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     6541 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     6601 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     6661 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     6721 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     6781 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     6841 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     6901 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     6961 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     7021 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     7081 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     7141 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     7201 ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg
     7261 ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga
     7321 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa
     7381 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg
     7441 ttgacattga ttattgacta ggcttttgca aaaagctttg caaagatgga taaagtttta
     7501 aacagagagg aatctttgca gctaatggac cttctaggtc ttgaaaggag tgggaatt
//
