LOCUS       sequence-377226-        7891 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 7891)
  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 7891)
  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: TOE1
FEATURES             Location/Qualifiers
     source          1..7891
                     /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(2848..2872)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2997..3038
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3493..3510)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3499..3723
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3856..3875)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4007..4054
                     /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             4062..5084
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4062..5084
                     /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    5214..5335
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5251..5270)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5305..5324
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5384..5400)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5384..5400)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5397..5419)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5408..5424
                     /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(5432..5438,5439..5456,5457..5462))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5477..5498
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5615..5632)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5786..6374)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5866..5885)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6545..7336,7337..7405))
                     /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     7168..7187
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7406..7510)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7585..7604)
                     /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 ccgccgacag tgacgatggc gcagtttcag ctcccgcagc ttccgacggt ggtgtcagca
     1381 aaagcacaac atctggggag gagctagtag tccaggttcc cgtagtggat gtgcaaagca
     1441 acaacttcaa ggagatgtgg ccatccctcc tgctagccat aaagacagct aatttcgtgg
     1501 ctgtggacac ggagctgagt gggcttgggg acaggaagag tttgctgaac cagtgcattg
     1561 aggaacgtta caaggccgtg tgtcatgctg ccaggacccg ttctatcctt tccctgggcc
     1621 tcgcctgctt caagcggcag ccagacaagg gtgaacattc ctatctggct caagtgttca
     1681 atctcactct gctgtgcatg gaggagtatg tcatagaacc aaagtctgtg cagttcctga
     1741 tacagcatgg cttcaacttc aaccagcagt atgcccaagg catcccctac cataagggca
     1801 atgacaaggg tgatgagagc cagagccagt cagtacggac cctattcctg gagctaatcc
     1861 gagcccgccg gcccctggtg ctacacaatg gccttataga cttggtgttc ctgtaccaga
     1921 acttctatgc acacctccct gagagtctgg gaaccttcac cgctgacctg tgtgagatgt
     1981 tcccagcagg catttatgac accaaatatg ctgctgagtt tcatgcccgt ttcgtggcct
     2041 cctacttaga atatgccttc cggaaatgtg aacgggaaaa tgggaagcag cgggcagctg
     2101 gcagcccaca ccttaccctg gagttctgca actatccttc cagcatgagg gaccatattg
     2161 attaccgctg ctgcctgccc ccagcaaccc accgtcctca tcccaccagc atctgtgaca
     2221 acttctcggc ttatggctgg tgccccctgg gaccacagtg tcctcagtct cacgatattg
     2281 accttatcat tgacactgat gaggctgcgg cagaggacaa gcggcgacgg cgacgacgta
     2341 gggaaaaacg gaagagggct ttattgaacc taccggggac acagacctct ggggaagcta
     2401 aggatggtcc tcccaagaag caggtctgtg gggatagcat caagcctgaa gaaaccgagc
     2461 aggaggtggc tgccgatgaa actaggaacc tgcctcactc caagcaaggc aacaaaaatg
     2521 acttagagat ggggattaag gcagcaaggc ctgaaatagc tgatagagct acctcagaag
     2581 tgccagggag ccaagccagt cctaacccag tgcctgggga tggattgcac cgggctggtt
     2641 ttgatgcctt tatgacaggt tatgtgatgg cctatgtgga agtgagccag ggaccgcagc
     2701 cctgcagctc tggaccctgg ctccctgaat gccacaataa ggtatatttg agtggcaaag
     2761 ctgtacccct cacagtggcc aagagccagt tctctcgttc ctccaaagcc cacaatcaga
     2821 agatgaagct cacttggggc agtagctgcc caactttctt gtacaaagtg gttgatatcc
     2881 agcacagtgg cggccgctcg agtctagagg gcccgcggtt cgaaaccagc ggaggcggcg
     2941 gaagcggggg aggaggcagc ggcggaggcg gcagcggggg cggcggaagc gctaccggta
     3001 agcctatccc taaccctctc ctcggtctcg attctacgcg taccggtatg gcttctaact
     3061 ttactcagtt cgttctcgtc gacaatggcg gaactggcga cgtgactgtc gccccaagca
     3121 acttcgctaa cggggtcgct gaatggatca gctctaactc gcgatcacag gcttacaaag
     3181 taacctgtag cgttcgtcag agctctgcgc agaatcgcaa atacaccatc aaagtcgagg
     3241 tgcctaaagt ggcaacccag actgttggtg gtgaagagct tcctgtagcc ggatggagat
     3301 cttacttaaa tatggaacta accattccaa ttttcgccac gaattccgac tgcgagctta
     3361 ttgttaaggc aatgcaaggt ctcctaaaag atggaaaccc gattccctcg gccatcgcgg
     3421 ccaactccgg catctacgga ggtggaggtg gatctcgtac gggttagtaa tgagtttaaa
     3481 cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     3541 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     3601 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     3661 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     3721 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
     3781 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     3841 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     3901 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     3961 acctcgaccc caaaaaactt gattagggtg atggttcacg tacctagaag ttcctattcc
     4021 gaagttccta ttctctagaa agtataggaa cttccttggc caaaaagcct gaactcaccg
     4081 cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc
     4141 tctcggaggg cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc
     4201 tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg
     4261 catcggccgc gctcccgatt ccggaagtgc ttgacattgg ggaattcagc gagagcctga
     4321 cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac
     4381 tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta
     4441 gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc
     4501 gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg
     4561 acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact
     4621 gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca
     4681 atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg
     4741 aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct
     4801 acttcgagcg gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc
     4861 gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt
     4921 gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac
     4981 aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata
     5041 gtggaaaccg acgccccagc actcgtccga gggcaaagga atagcacgta ctacgagatt
     5101 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     5161 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     5221 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     5281 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     5341 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     5401 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     5461 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     5521 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     5581 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     5641 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     5701 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     5761 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     5821 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     5881 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     5941 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     6001 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     6061 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     6121 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     6181 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     6241 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     6301 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     6361 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     6421 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     6481 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     6541 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     6601 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     6661 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     6721 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     6781 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     6841 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     6901 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     6961 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     7021 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     7081 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     7141 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     7201 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     7261 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     7321 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     7381 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     7441 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     7501 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc
     7561 cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     7621 tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac
     7681 aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc
     7741 gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctaggctttt
     7801 gcaaaaagct ttgcaaagat ggataaagtt ttaaacagag aggaatcttt gcagctaatg
     7861 gaccttctag gtcttgaaag gagtgggaat t
//
