LOCUS       sequence-376750-        8395 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 8395)
  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 8395)
  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: DHX58
FEATURES             Location/Qualifiers
     source          1..8395
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        119..1297
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          350..1288
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1245..1265
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     1314..1333
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1314..1332
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1411..1435
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(3470..3494)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3619..3660
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(4115..4132)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4121..4345
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4478..4497)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4629..4676
                     /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             4684..5706
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4684..5706
                     /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    5836..5957
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5873..5892)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5927..5946
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6006..6022)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6006..6022)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6019..6041)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6030..6046
                     /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(6054..6060,6061..6078,6079..6084))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6099..6120
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6237..6254)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6408..6996)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6488..6507)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7167..7958,7959..8027))
                     /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     7790..7809
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8028..8132)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8207..8226)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ttgacattga ttattgacta ggcttttgca aaaagctttg caaagatgga taaagtttta
       61 aacagagagg aatctttgca gctaatggac cttctaggtc ttgaaaggag tgggaattgg
      121 ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg
      181 aggggtcggc aattgaaccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga
      241 tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt
      301 agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg taagtgccgt
      361 gtgtggttcc cgcgggcctg gcctctttac gggttatggc ccttgcgtgc cttgaattac
      421 ttccacctgg ctgcagtacg tgattcttga tcccgagctt cgggttggaa gtgggtggga
      481 gagttcgagg ccttgcgctt aaggagcccc ttcgcctcgt gcttgagttg aggcctggcc
      541 tgggcgctgg ggccgccgcg tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt
      601 cgataagtct ctagccattt aaaatttttg atgacctgct gcgacgcttt ttttctggca
      661 agatagtctt gtaaatgcgg gccaagatct gcacactggt atttcggttt ttggggccgc
      721 gggcggcgac ggggcccgtg cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc
      781 gcggccaccg agaatcggac gggggtagtc tcaagctggc cggcctgctc tggtgcctgg
      841 cctcgcgccg ccgtgtatcg ccccgccctg ggcggcaagg ctggcccggt cggcaccagt
      901 tgcgtgagcg gaaagatggc cgcttcccgg ccctgctgca gggagctcaa aatggaggac
      961 gcggcgctcg ggagagcggg cgggtgagtc acccacacaa aggaaaaggg cctttccgtc
     1021 ctcagccgtc gcttcatgtg actccacgga gtaccgggcg ccgtccaggc acctcgatta
     1081 gttctcgagc ttttggagta cgtcgtcttt aggttggggg gaggggtttt atgcgatgga
     1141 gtttccccac actgagtggg tggagactga agttaggcca gcttggcact tgatgtaatt
     1201 ctccttggaa tttgcccttt ttgagtttgg atcttggttc attctcaagc ctcagacagt
     1261 ggttcaaagt ttttttcttc catttcaggt gtcgtgagga attagcttgg tactaatacg
     1321 actcactata gggagaccca agctggctag gtaagcttgg taccgagctc ggatccacta
     1381 gtccagtgtg gtggaattct gcagatatca acaagtttgt acaaaaaagt tggcatggag
     1441 cttcggtcct accaatggga ggtgatcatg cctgccctgg agggcaagaa tatcatcatc
     1501 tggctgccca cgggtgccgg gaagacccgg gcggctgctt atgtggccaa gcggcaccta
     1561 gagactgtgg atggagccaa ggtggttgta ttggtcaaca gggtgcacct ggtgacccag
     1621 catggtgaag agttcaggcg catgctggat ggacgctgga ccgtgacaac cctgagtggg
     1681 gacatgggac cacgtgctgg ctttggccac ctggcccggt gccatgacct gctcatctgc
     1741 acagcagagc ttctgcagat ggcactgacc agccccgagg aggaggagca cgtggagctc
     1801 actgtcttct ccctgatcgt ggtggatgag tgccaccaca cgcacaagga caccgtctac
     1861 aacgtcatca tgagccagta cctagaactt aaactccaga gggcacagcc gctaccccag
     1921 gtgctgggtc tcacagcctc cccaggcact ggcggggcct ccaaactcga tggggccatc
     1981 aaccacgtcc tgcagctctg tgccaacttg gacacgtggt gcatcatgtc accccagaac
     2041 tgctgccccc agctgcagga gcacagccaa cagccttgca aacagtacaa cctctgccac
     2101 aggcgcagcc aggatccgtt tggggacttg ctgaagaagc tcatggacca aatccatgac
     2161 cacctggaga tgcctgagtt gagccggaaa tttgggacgc aaatgtatga gcagcaggtg
     2221 gtgaagctga gtgaggctgc ggctttggct gggcttcagg agcaacgggt gtatgcgctt
     2281 cacctgaggc gctacaatga cgcgctgctc atccatgaca ccgtccgcgc cgtggatgcc
     2341 ttggctgcgc tgcaggattt ctatcacagg gagcacgtca ctaaaaccca gatcctgtgt
     2401 gccgagcgcc ggctgctggc cctgttcgat gaccgcaaga atgagctggc ccacttggca
     2461 actcatggcc cagagaatcc aaaactggag atgctggaaa agatcctgca aaggcagttc
     2521 agtagctcta acagccctcg gggtatcatc ttcacccgca cccgccaaag cgcacactcc
     2581 ctcctgctct ggctccagca gcagcagggc ctgcagactg tggacatccg ggcccagcta
     2641 ctgattgggg ctgggaacag cagccagagc acccacatga cccagaggga ccagcaagaa
     2701 gtgatccaga agttccaaga tggaaccctg aaccttctgg tggccacgag tgtggcggag
     2761 gaggggctgg acatcccaca ttgcaatgtg gtggtgcgtt atgggctctt gaccaatgaa
     2821 atctccatgg tccaggccag gggccgtgcc cgggccgatc agagtgtata cgcgtttgta
     2881 gcaactgaag gtagccggga gctgaagcgg gagctgatca acgaggcgct ggagacgctg
     2941 atggagcagg cagtggctgc tgtgcagaaa atggaccagg ccgagtacca ggccaagatc
     3001 cgggatctgc agcaggcagc cttgaccaag cgggcggccc aggcagccca gcgggagaac
     3061 cagcggcagc agttcccagt ggagcacgtg cagctactct gcatcaactg catggtggct
     3121 gtgggccatg gcagcgacct gcggaaggtg gagggcaccc accatgtcaa tgtgaacccc
     3181 aacttctcga actactataa tgtctccagg gatcctgtgg tcatcaacaa agtcttcaag
     3241 gactggaagc ctgggggtgt catcagctgc aggaactgtg gggaggtctg gggtctgcag
     3301 atgatctaca agtcagtgaa gctgccagtg ctcaaagtcc gcagcatgct gctggagacc
     3361 cctcaggggc ggatccaggc caaaaagtgg tcccgcgtgc ccttctccgt gcctgacttt
     3421 gacttcctgc agcattgtgc cgagaacttg tcggacctct ccctggactg cccaactttc
     3481 ttgtacaaag tggttgatat ccagcacagt ggcggccgct cgagtctaga gggcccgcgg
     3541 ttcgaaacca gcggaggcgg cggaagcggg ggaggaggca gcggcggagg cggcagcggg
     3601 ggcggcggaa gcgctaccgg taagcctatc cctaaccctc tcctcggtct cgattctacg
     3661 cgtaccggta tggcttctaa ctttactcag ttcgttctcg tcgacaatgg cggaactggc
     3721 gacgtgactg tcgccccaag caacttcgct aacggggtcg ctgaatggat cagctctaac
     3781 tcgcgatcac aggcttacaa agtaacctgt agcgttcgtc agagctctgc gcagaatcgc
     3841 aaatacacca tcaaagtcga ggtgcctaaa gtggcaaccc agactgttgg tggtgaagag
     3901 cttcctgtag ccggatggag atcttactta aatatggaac taaccattcc aattttcgcc
     3961 acgaattccg actgcgagct tattgttaag gcaatgcaag gtctcctaaa agatggaaac
     4021 ccgattccct cggccatcgc ggccaactcc ggcatctacg gaggtggagg tggatctcgt
     4081 acgggttagt aatgagttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     4141 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     4201 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     4261 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     4321 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     4381 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     4441 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     4501 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     4561 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     4621 cgtacctaga agttcctatt ccgaagttcc tattctctag aaagtatagg aacttccttg
     4681 gccaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac
     4741 agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat
     4801 gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat
     4861 cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt
     4921 ggggaattca gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg
     4981 caagacctgc ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat
     5041 gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga
     5101 atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat
     5161 cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag
     5221 ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc
     5281 tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg
     5341 atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct
     5401 tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg
     5461 cggctccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac
     5521 ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga
     5581 gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc
     5641 tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag
     5701 gaatagcacg tactacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     5761 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     5821 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     5881 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     5941 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa
     6001 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     6061 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     6121 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     6181 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     6241 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     6301 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     6361 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     6421 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     6481 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     6541 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     6601 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     6661 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     6721 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     6781 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     6841 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     6901 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     6961 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     7021 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     7081 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     7141 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     7201 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     7261 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     7321 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     7381 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     7441 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     7501 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     7561 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     7621 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     7681 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     7741 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     7801 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     7861 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     7921 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     7981 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     8041 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     8101 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     8161 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
     8221 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
     8281 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
     8341 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcg
//
