LOCUS       sequence-377224-        7399 bp    DNA     circular SYN 15-APR-2025
DEFINITION  For use in RBP tethering screen.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7399)
  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 7399)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: TOB2
FEATURES             Location/Qualifiers
     source          1..7399
                     /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(2356..2380)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2505..2546
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3001..3018)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3007..3231
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3364..3383)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3515..3562
                     /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             3570..4592
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3570..4592
                     /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    4722..4843
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4759..4778)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4813..4832
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4892..4908)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4892..4908)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4905..4927)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4916..4932
                     /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(4940..4946,4947..4964,4965..4970))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4985..5006
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5123..5140)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5294..5882)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5374..5393)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6053..6844,6845..6913))
                     /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     6676..6695
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6914..7018)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7093..7112)
                     /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 gcaggctcca
     1321 ccatgcagct agagatcaaa gtggccctga acttcatcat ctcctacttg tacaacaagc
     1381 tgccccggcg ccgggcagac ctgtttgggg aggagctaga gcggcttttg aaaaagaaat
     1441 atgaaggcca ctggtaccct gagaagccac tgaaaggctc tggcttccgc tgtgttcaca
     1501 ttggggagat ggtggacccc gtggtggagc tggccgccaa gcggagtggc ctggcagtgg
     1561 aagatgtgcg ggccaatgtg cctgaggagc tgagtgtctg gattgatccc tttgaggtgt
     1621 cctaccagat tggtgagaag ggagctgtga aagtgctgta cctggatgac agtgagggtt
     1681 gcggtgcccc agagctggac aaggagatca agagcagctt caaccctgac gcccaggtgt
     1741 tcgtgcccat tggcagccag gacagctccc tgtccaactc cccatcgcca tcctttggcc
     1801 agtcacccag ccctaccttc attccccgct ccgctcagcc catcaccttc accaccgcct
     1861 ccttcgctgc caccaaattt ggctccacta agatgaagaa ggggggcggg gcagcaagtg
     1921 gtgggggtgt agccagcagt ggggcgggtg gccagcagcc accacagcag cctcgcatgg
     1981 cccgctcacc caccaacagc ctgctgaagc acaagagcct ctctctgtct atgcattcac
     2041 tgaacttcat cacggccaac ccggcccctc agtcccagct ctcacccaat gccaaggagt
     2101 tcgtgtacaa cggtggtggc tcacccagcc tcttctttga tgcggccgat ggccagggca
     2161 gcggcacccc aggcccgttt ggaggcagtg gggctggcac ctgcaacagc agcagctttg
     2221 acatggccca ggtatttgga ggtggtgcca acagcctctt cctggagaag acaccctttg
     2281 tggaaggcct cagctacaac ctgaacacca tgcagtatcc cagccagcag ttccagcccg
     2341 tggtgctggc caacgaccca gctttcttgt acaaagtggt tgatatccag cacagtggcg
     2401 gccgctcgag tctagagggc ccgcggttcg aaaccagcgg aggcggcgga agcgggggag
     2461 gaggcagcgg cggaggcggc agcgggggcg gcggaagcgc taccggtaag cctatcccta
     2521 accctctcct cggtctcgat tctacgcgta ccggtatggc ttctaacttt actcagttcg
     2581 ttctcgtcga caatggcgga actggcgacg tgactgtcgc cccaagcaac ttcgctaacg
     2641 gggtcgctga atggatcagc tctaactcgc gatcacaggc ttacaaagta acctgtagcg
     2701 ttcgtcagag ctctgcgcag aatcgcaaat acaccatcaa agtcgaggtg cctaaagtgg
     2761 caacccagac tgttggtggt gaagagcttc ctgtagccgg atggagatct tacttaaata
     2821 tggaactaac cattccaatt ttcgccacga attccgactg cgagcttatt gttaaggcaa
     2881 tgcaaggtct cctaaaagat ggaaacccga ttccctcggc catcgcggcc aactccggca
     2941 tctacggagg tggaggtgga tctcgtacgg gttagtaatg agtttaaacc cgctgatcag
     3001 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     3061 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     3121 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     3181 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     3241 cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa
     3301 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     3361 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     3421 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     3481 aaaaacttga ttagggtgat ggttcacgta cctagaagtt cctattccga agttcctatt
     3541 ctctagaaag tataggaact tccttggcca aaaagcctga actcaccgcg acgtctgtcg
     3601 agaagtttct gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg
     3661 aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata
     3721 gctgcgccga tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc
     3781 tcccgattcc ggaagtgctt gacattgggg aattcagcga gagcctgacc tattgcatct
     3841 cccgccgtgc acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc
     3901 tgcagccggt cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg
     3961 ggttcggccc attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat
     4021 gcgcgattgc tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg
     4081 cgtccgtcgc gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc
     4141 ggcacctcgt gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa
     4201 cagcggtcat tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca
     4261 tcttcttctg gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga
     4321 ggcatccgga gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg
     4381 accaactcta tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc
     4441 gatgcgacgc aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca
     4501 gaagcgcggc cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac
     4561 gccccagcac tcgtccgagg gcaaaggaat agcacgtact acgagatttc gattccaccg
     4621 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     4681 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt
     4741 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     4801 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
     4861 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     4921 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     4981 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     5041 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     5101 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     5161 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     5221 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     5281 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     5341 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     5401 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     5461 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     5521 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     5581 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     5641 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     5701 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     5761 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5821 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     5881 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     5941 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     6001 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     6061 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     6121 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     6181 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     6241 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     6301 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     6361 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     6421 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     6481 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     6541 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     6601 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     6661 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     6721 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     6781 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     6841 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     6901 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     6961 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     7021 catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg
     7081 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc
     7141 ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg
     7201 cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga
     7261 tgtacgggcc agatatacgc gttgacattg attattgact aggcttttgc aaaaagcttt
     7321 gcaaagatgg ataaagtttt aaacagagag gaatctttgc agctaatgga ccttctaggt
     7381 cttgaaagga gtgggaatt
//
