LOCUS       sequence-375893-        7568 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 7568)
  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 7568)
  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: ADD1
FEATURES             Location/Qualifiers
     source          1..7568
                     /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(2525..2549)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2674..2715
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3170..3187)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3176..3400
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3533..3552)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3684..3731
                     /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             3739..4761
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3739..4761
                     /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    4891..5012
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4928..4947)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4982..5001
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5061..5077)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5061..5077)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5074..5096)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5085..5101
                     /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(5109..5115,5116..5133,5134..5139))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5154..5175
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5292..5309)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5463..6051)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5543..5562)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6222..7013,7014..7082))
                     /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     6845..6864
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7083..7187)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7262..7281)
                     /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 gcaggctgcc
     1321 accatgaatg gtgattctcg tgctgcggtg gtgacctcac cacccccgac cacagcccct
     1381 cacaaggaga ggtacttcga ccgagtagat gagaacaacc cagagtactt gagggagagg
     1441 aacatggcac cagaccttcg ccaggacttc aacatgatgg agcaaaagaa gagggtgtcc
     1501 atgattctgc aaagccctgc tttctgtgaa gaattggaat caatgataca ggagcaattt
     1561 aagaagggga agaaccccac aggcctattg gcattacagc agattgcaga ttttatgacc
     1621 acgaatgtac caaatgtcta cccagcagct ccgcaaggag ggatggctgc cttaaacatg
     1681 agtcttggta tggtgactcc tgtgaacgat cttagaggat ctgattctat tgcgtatgac
     1741 aaaggagaga agttattacg gtgtaaattg gcagcgtttt atagactagc agatctcttt
     1801 gggtggtctc agcttatcta caatcatatc acaaccagag tgaactccga gcaggaacac
     1861 ttcctcattg tcccttttgg gcttctttac agtgaagtga ctgcatccag tttggttaag
     1921 atcaatctac aaggagatat agtagatcgt ggaagcacta atctgggagt gaatcaggcc
     1981 ggcttcacct tacactctgc aatttatgct gcacgcccgg acgtgaagtg cgtcgtgcac
     2041 attcacaccc cagcaggggc tgcggtctct gcaatgaaat gtggcctctt gccaatctcc
     2101 ccggaggcgc tttcccttgg agaagtggct tatcatgact accatggcat tctggttgat
     2161 gaagaggaaa aagttttgat tcagaaaaat ctggggccta aaagcaaggt tcttattctc
     2221 cggaaccatg ggctcgtgtc agttggagag agcgttgagg aggccttcta ttacatccat
     2281 aaccttgtgg ttgcctgtga gatccaggtt cgaactctgg ccagtgcagg aggaccagac
     2341 aacttagtcc tgctgaatcc tgagaagtac aaagccaagt cccgttcccc agggtctccg
     2401 gtaggggaag gcactggatc gcctcccaag tggcagattg gtgagcagga atttgaagcc
     2461 ctcatgcgga tgctcgataa tctgggctac agaactagca gcagcgggag aagacaagat
     2521 ggcgacccag ctttcttgta caaagtggtt gatatccagc acagtggcgg ccgctcgagt
     2581 ctagagggcc cgcggttcga aaccagcgga ggcggcggaa gcgggggagg aggcagcggc
     2641 ggaggcggca gcgggggcgg cggaagcgct accggtaagc ctatccctaa ccctctcctc
     2701 ggtctcgatt ctacgcgtac cggtatggct tctaacttta ctcagttcgt tctcgtcgac
     2761 aatggcggaa ctggcgacgt gactgtcgcc ccaagcaact tcgctaacgg ggtcgctgaa
     2821 tggatcagct ctaactcgcg atcacaggct tacaaagtaa cctgtagcgt tcgtcagagc
     2881 tctgcgcaga atcgcaaata caccatcaaa gtcgaggtgc ctaaagtggc aacccagact
     2941 gttggtggtg aagagcttcc tgtagccgga tggagatctt acttaaatat ggaactaacc
     3001 attccaattt tcgccacgaa ttccgactgc gagcttattg ttaaggcaat gcaaggtctc
     3061 ctaaaagatg gaaacccgat tccctcggcc atcgcggcca actccggcat ctacggaggt
     3121 ggaggtggat ctcgtacggg ttagtaatga gtttaaaccc gctgatcagc ctcgactgtg
     3181 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     3241 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     3301 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     3361 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
     3421 agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
     3481 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     3541 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     3601 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     3661 tagggtgatg gttcacgtac ctagaagttc ctattccgaa gttcctattc tctagaaagt
     3721 ataggaactt ccttggccaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg
     3781 atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt
     3841 gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat
     3901 ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg
     3961 gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca
     4021 cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc
     4081 gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca
     4141 ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct
     4201 gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg
     4261 caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg
     4321 cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt
     4381 gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg
     4441 aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag
     4501 cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat
     4561 cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca
     4621 atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc
     4681 gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact
     4741 cgtccgaggg caaaggaata gcacgtacta cgagatttcg attccaccgc cgccttctat
     4801 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     4861 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     4921 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     4981 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     5041 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     5101 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     5161 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     5221 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     5281 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     5341 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     5401 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     5461 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     5521 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     5581 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     5641 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     5701 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     5761 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     5821 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     5881 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     5941 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     6001 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     6061 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     6121 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     6181 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     6241 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     6301 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     6361 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     6421 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     6481 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     6541 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     6601 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     6661 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     6721 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     6781 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     6841 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     6901 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     6961 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     7021 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     7081 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     7141 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     7201 aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca
     7261 gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg
     7321 aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca
     7381 attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca
     7441 gatatacgcg ttgacattga ttattgacta ggcttttgca aaaagctttg caaagatgga
     7501 taaagtttta aacagagagg aatctttgca gctaatggac cttctaggtc ttgaaaggag
     7561 tgggaatt
//
