LOCUS       sequence-375991-        7699 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 7699)
  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 7699)
  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: SFRS17A
FEATURES             Location/Qualifiers
     source          1..7699
                     /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(2656..2680)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2805..2846
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3301..3318)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3307..3531
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3664..3683)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3815..3862
                     /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             3870..4892
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3870..4892
                     /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    5022..5143
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5059..5078)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5113..5132
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5192..5208)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5192..5208)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5205..5227)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5216..5232
                     /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(5240..5246,5247..5264,5265..5270))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5285..5306
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5423..5440)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5594..6182)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5674..5693)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6353..7144,7145..7213))
                     /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     6976..6995
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7214..7318)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7393..7412)
                     /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 cagcggctac catcgtgcac gacacgtctg aggccgtgga gctctgccct gcttacggct
     1381 tgtacctgaa gcccatcacc aagatgacca tcagcgtggc actcccgcag ctgaagcagc
     1441 cggggaagtc catctccaac tgggaggtga tggagaggct gaagggcatg gtgcagaacc
     1501 accagttctc cacgctgcgt atttccaaga gcaccatgga cttcatccgc ttcgaggggg
     1561 aggtggagaa caagagcctg gtcaagtctt ttctggcctg cctggacggc aagaccatca
     1621 agctcagcgg cttctccgac atcctgaagg tgcgcgcggc cgagttcaag atcgacttcc
     1681 ccacccgcca cgactgggac tccttcttcc gcgacgccaa ggacatgaac gagaccctgc
     1741 cgggggagcg gccggacacc atccacctgg aggggctgcc ctgcaagtgg ttcgccctga
     1801 aggagtcggg ctccgagaag cccagcgagg acgtcctggt caaggtgttt gagaagttcg
     1861 gggagatccg gaatgtggac atccccatgc tggactccta ccgggaggag atgacgggcc
     1921 gcaacttcca caccttcagt ttcggggggc acttgaactt cgaggcctat gtgcagtacc
     1981 gcgagtacat gggcttcatc caggccatga gcgccctgcg cgggatgaaa ctcatgtaca
     2041 agggcgagga cggcaaggcc gtggcctgca acatcaaggt ttcttttgat tcgaccaaac
     2101 acctgagtga tgcctcaatt aagaagcggc agctggagag gcagaagctt caggaactgg
     2161 agcagcaaag agaagaacaa aagcgcagag agaaggaagc ggaggagagg cagcgagcgg
     2221 aggaaaggaa acaaaaggag ctggaagagc tggagcgaga gaggaaaaga gaagagaagc
     2281 ttcgcaagag ggagcagaag cagagggacc gtgagctgcg ccggaatcag aagaagctgg
     2341 agaagctgca ggcggaggag cagaagcagc tgcaggagaa gatcaagctg gaggagcgca
     2401 agctgctgct ggcccagagg aacctgcagt ccatccggct catcgccgag ctgctcagca
     2461 gagccaaggt acccgggggc tccctctgca gccgccagcc gcgcccgggc tgccctcagt
     2521 gccctcccct gaaatgcggg cggcgtcacg gcgccgtttc cccgccggct gcagctgtag
     2581 ctacgaaacc agctttaatg ccgaggatga cggctccttc ccgggagggt gtagcgctcg
     2641 tctgtcgttc ccgatgccca actttcttgt acaaagtggt tgatatccag cacagtggcg
     2701 gccgctcgag tctagagggc ccgcggttcg aaaccagcgg aggcggcgga agcgggggag
     2761 gaggcagcgg cggaggcggc agcgggggcg gcggaagcgc taccggtaag cctatcccta
     2821 accctctcct cggtctcgat tctacgcgta ccggtatggc ttctaacttt actcagttcg
     2881 ttctcgtcga caatggcgga actggcgacg tgactgtcgc cccaagcaac ttcgctaacg
     2941 gggtcgctga atggatcagc tctaactcgc gatcacaggc ttacaaagta acctgtagcg
     3001 ttcgtcagag ctctgcgcag aatcgcaaat acaccatcaa agtcgaggtg cctaaagtgg
     3061 caacccagac tgttggtggt gaagagcttc ctgtagccgg atggagatct tacttaaata
     3121 tggaactaac cattccaatt ttcgccacga attccgactg cgagcttatt gttaaggcaa
     3181 tgcaaggtct cctaaaagat ggaaacccga ttccctcggc catcgcggcc aactccggca
     3241 tctacggagg tggaggtgga tctcgtacgg gttagtaatg agtttaaacc cgctgatcag
     3301 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     3361 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     3421 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     3481 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     3541 cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa
     3601 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     3661 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     3721 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     3781 aaaaacttga ttagggtgat ggttcacgta cctagaagtt cctattccga agttcctatt
     3841 ctctagaaag tataggaact tccttggcca aaaagcctga actcaccgcg acgtctgtcg
     3901 agaagtttct gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg
     3961 aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata
     4021 gctgcgccga tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc
     4081 tcccgattcc ggaagtgctt gacattgggg aattcagcga gagcctgacc tattgcatct
     4141 cccgccgtgc acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc
     4201 tgcagccggt cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg
     4261 ggttcggccc attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat
     4321 gcgcgattgc tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg
     4381 cgtccgtcgc gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc
     4441 ggcacctcgt gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa
     4501 cagcggtcat tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca
     4561 tcttcttctg gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga
     4621 ggcatccgga gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg
     4681 accaactcta tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc
     4741 gatgcgacgc aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca
     4801 gaagcgcggc cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac
     4861 gccccagcac tcgtccgagg gcaaaggaat agcacgtact acgagatttc gattccaccg
     4921 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     4981 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt
     5041 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     5101 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
     5161 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     5221 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     5281 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     5341 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     5401 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     5461 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     5521 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     5581 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     5641 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     5701 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     5761 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     5821 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     5881 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     5941 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     6001 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     6061 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     6121 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     6181 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     6241 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     6301 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     6361 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     6421 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     6481 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     6541 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     6601 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     6661 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     6721 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     6781 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     6841 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     6901 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     6961 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     7021 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     7081 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     7141 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     7201 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     7261 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     7321 catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg
     7381 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc
     7441 ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg
     7501 cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga
     7561 tgtacgggcc agatatacgc gttgacattg attattgact aggcttttgc aaaaagcttt
     7621 gcaaagatgg ataaagtttt aaacagagag gaatctttgc agctaatgga ccttctaggt
     7681 cttgaaagga gtgggaatt
//
