LOCUS       sequence-376032-        7663 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 7663)
  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 7663)
  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: CSTF1
FEATURES             Location/Qualifiers
     source          1..7663
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        56..1234
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          287..1225
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1182..1202
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     1251..1270
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1251..1269
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1348..1372
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2675..2699)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2824..2865
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3320..3337)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3326..3550
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3683..3702)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3834..3881
                     /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             3889..4911
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3889..4911
                     /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    5041..5162
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5078..5097)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5132..5151
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5211..5227)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5211..5227)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5224..5246)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5235..5251
                     /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(5259..5265,5266..5283,5284..5289))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5304..5325
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5442..5459)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5613..6201)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5693..5712)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6372..7163,7164..7232))
                     /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     6995..7014
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7233..7337)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7412..7431)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 agagaggaat ctttgcagct aatggacctt ctaggtcttg aaaggagtgg gaattggctc
       61 cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg
      121 ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt
      181 cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt
      241 cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa gtgccgtgtg
      301 tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt gaattacttc
      361 cacctggctg cagtacgtga ttcttgatcc cgagcttcgg gttggaagtg ggtgggagag
      421 ttcgaggcct tgcgcttaag gagccccttc gcctcgtgct tgagttgagg cctggcctgg
      481 gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc gcctgtctcg ctgctttcga
      541 taagtctcta gccatttaaa atttttgatg acctgctgcg acgctttttt tctggcaaga
      601 tagtcttgta aatgcgggcc aagatctgca cactggtatt tcggtttttg gggccgcggg
      661 cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc tgcgagcgcg
      721 gccaccgaga atcggacggg ggtagtctca agctggccgg cctgctctgg tgcctggcct
      781 cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg caccagttgc
      841 gtgagcggaa agatggccgc ttcccggccc tgctgcaggg agctcaaaat ggaggacgcg
      901 gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct ttccgtcctc
      961 agccgtcgct tcatgtgact ccacggagta ccgggcgccg tccaggcacc tcgattagtt
     1021 ctcgagcttt tggagtacgt cgtctttagg ttggggggag gggttttatg cgatggagtt
     1081 tccccacact gagtgggtgg agactgaagt taggccagct tggcacttga tgtaattctc
     1141 cttggaattt gccctttttg agtttggatc ttggttcatt ctcaagcctc agacagtggt
     1201 tcaaagtttt tttcttccat ttcaggtgtc gtgaggaatt agcttggtac taatacgact
     1261 cactataggg agacccaagc tggctaggta agcttggtac cgagctcgga tccactagtc
     1321 cagtgtggtg gaattctgca gatatcaaca agtttgtaca aaaaagcagg ctccaccatg
     1381 tacagaacca aagtgggctt gaaggaccgc cagcagctct acaagctgat cattagccag
     1441 ctgctatatg acggctacat cagcatcgcc aatggcctca tcaatgaaat caagcctcag
     1501 tctgtgtgtg caccctcgga gcagctcctg catctcatca aactcggaat ggaaaacgat
     1561 gacaccgcag ttcagtatgc aattggtcgt tcagatactg ttgcccctgg cacagggatt
     1621 gacctggaat ttgatgcaga tgttcagact atgtccccag aggcttctga gtacgaaaca
     1681 tgctatgtca catcacataa aggaccatgc cgtgtagcta cctatagtag agatggacag
     1741 ttaatagcta ctgggtctgc tgatgcttcg ataaagatac ttgacacaga gaggatgttg
     1801 gccaaaagtg ccatgccaat agaggtcatg atgaatgaga ccgcacaaca aaatatggaa
     1861 aaccacccag tgattcgaac tctttatgac catgtggatg aagtcacgtg ccttgctttc
     1921 cacccaacag aacagatcct ggcttctggt tcaagggatt atactcttaa attatttgat
     1981 tattccaaac catcagcaaa aagagccttc aaatacattc aggaagctga aatgttacgt
     2041 tccatctctt ttcatccttc tggagacttt atacttgtcg gaactcagca tcctactctt
     2101 cgcctttatg atatcaacac ctttcaatgt tttgtctctt gcaatcctca agatcaacac
     2161 accgatgcta tatgttccgt taattacaat tctagtgcca atatgtacgt aactggaagc
     2221 aaggacggct gcatcaaatt atgggatggt gtttcaaatc gatgcatcac aacttttgag
     2281 aaagcacatg acggtgctga agtttgttct gccatttttt ccaaaaattc taaatacatt
     2341 ctctcaagtg gaaaagactc tgtagctaaa ctttgggaaa tatcaacggg acgaacactg
     2401 gtcagataca cgggcgcggg tttaagtgga cgccaggtgc accggacaca ggctgtgttt
     2461 aaccacaccg aggactatgt gttgctgccc gacgagagga cgatcagtct ttgctgctgg
     2521 gactcgagga cagccgagcg gagaaacctg ctgtcgttgg ggcacaacaa tattgtacgc
     2581 tgcatagtgc actcccccac caaccccggg ttcatgacgt gcagcgatga cttcagagcg
     2641 cggttttggt accggagatc gaccactgac ttggacccag ctttcttgta caaagtggtt
     2701 gatatccagc acagtggcgg ccgctcgagt ctagagggcc cgcggttcga aaccagcgga
     2761 ggcggcggaa gcgggggagg aggcagcggc ggaggcggca gcgggggcgg cggaagcgct
     2821 accggtaagc ctatccctaa ccctctcctc ggtctcgatt ctacgcgtac cggtatggct
     2881 tctaacttta ctcagttcgt tctcgtcgac aatggcggaa ctggcgacgt gactgtcgcc
     2941 ccaagcaact tcgctaacgg ggtcgctgaa tggatcagct ctaactcgcg atcacaggct
     3001 tacaaagtaa cctgtagcgt tcgtcagagc tctgcgcaga atcgcaaata caccatcaaa
     3061 gtcgaggtgc ctaaagtggc aacccagact gttggtggtg aagagcttcc tgtagccgga
     3121 tggagatctt acttaaatat ggaactaacc attccaattt tcgccacgaa ttccgactgc
     3181 gagcttattg ttaaggcaat gcaaggtctc ctaaaagatg gaaacccgat tccctcggcc
     3241 atcgcggcca actccggcat ctacggaggt ggaggtggat ctcgtacggg ttagtaatga
     3301 gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     3361 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     3421 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     3481 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     3541 ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg
     3601 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     3661 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     3721 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     3781 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtac ctagaagttc
     3841 ctattccgaa gttcctattc tctagaaagt ataggaactt ccttggccaa aaagcctgaa
     3901 ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg
     3961 atgcagctct cggagggcga agaatctcgt gctttcagct tcgatgtagg agggcgtgga
     4021 tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg
     4081 cactttgcat cggccgcgct cccgattccg gaagtgcttg acattgggga attcagcgag
     4141 agcctgacct attgcatctc ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa
     4201 accgaactgc ccgctgttct gcagccggtc gcggaggcca tggatgcgat cgctgcggcc
     4261 gatcttagcc agacgagcgg gttcggccca ttcggaccgc aaggaatcgg tcaatacact
     4321 acatggcgtg atttcatatg cgcgattgct gatccccatg tgtatcactg gcaaactgtg
     4381 atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat gctttgggcc
     4441 gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa caatgtcctg
     4501 acggacaatg gccgcataac agcggtcatt gactggagcg aggcgatgtt cggggattcc
     4561 caatacgagg tcgccaacat cttcttctgg aggccgtggt tggcttgtat ggagcagcag
     4621 acgcgctact tcgagcggag gcatccggag cttgcaggat cgccgcggct ccgggcgtat
     4681 atgctccgca ttggtcttga ccaactctat cagagcttgg ttgacggcaa tttcgatgat
     4741 gcagcttggg cgcagggtcg atgcgacgca atcgtccgat ccggagccgg gactgtcggg
     4801 cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt agaagtactc
     4861 gccgatagtg gaaaccgacg ccccagcact cgtccgaggg caaaggaata gcacgtacta
     4921 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     4981 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
     5041 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     5101 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     5161 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
     5221 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     5281 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     5341 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     5401 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     5461 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     5521 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     5581 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     5641 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     5701 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     5761 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     5821 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     5881 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     5941 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     6001 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     6061 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     6121 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     6181 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     6241 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     6301 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     6361 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     6421 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     6481 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     6541 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     6601 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     6661 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     6721 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     6781 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     6841 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     6901 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     6961 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     7021 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     7081 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     7141 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     7201 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     7261 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     7321 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga
     7381 gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     7441 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt
     7501 aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc
     7561 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta
     7621 ggcttttgca aaaagctttg caaagatgga taaagtttta aac
//
