LOCUS       sequence-377211-        8258 bp    DNA     circular SYN 14-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 8258)
  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 8258)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: DMPK
FEATURES             Location/Qualifiers
     source          1..8258
                     /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"
     RBS             1506..1514
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     protein_bind    complement(3215..3239)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3364..3405
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3860..3877)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3866..4090
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4223..4242)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4374..4421
                     /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             4429..5451
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4429..5451
                     /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    5581..5702
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5618..5637)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5672..5691
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5751..5767)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5751..5767)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5764..5786)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5775..5791
                     /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(5799..5805,5806..5823,5824..5829))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5844..5865
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5982..5999)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6153..6741)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6233..6252)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6912..7703,7704..7772))
                     /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     7535..7554
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7773..7877)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7952..7971)
                     /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 accatgtcag ccgaggtgcg gctgaggcgg ctccagcagc tggtgttgga cccgggcttc
     1381 ctggggctgg agcccctgct cgaccttctc ctgggcgtcc accaggagct gggcgcctcc
     1441 gaactggccc aggacaagta cgtggccgac ttcttgcagt gggcggagcc catcgtggtg
     1501 aggcttaagg aggtccgact gcagagggac gacttcgaga ttctgaaggt gatcggacgc
     1561 ggggcgttca gcgaggtagc ggtagtgaag atgaagcaga cgggccaggt gtatgccatg
     1621 aagatcatga acaagtggga catgctgaag aggggcgagg tgtcgtgctt ccgtgaggag
     1681 agggacgtgt tggtgaatgg ggaccggcgg tggatcacgc agctgcactt cgccttccag
     1741 gatgagaact acctgtacct ggtcatggag tattacgtgg gcggggacct gctgacactg
     1801 ctgagcaagt ttggggagcg gattccggcc gagatggcgc gcttctacct ggcggagatt
     1861 gtcatggcca tagactcggt gcaccggctt ggctacgtgc acagggacat caaacccgac
     1921 aacatcctgc tggaccgctg tggccacatc cgcctggccg acttcggctc ttgcctcaag
     1981 ctgcgggcag atggaacggt gcggtcgctg gtggctgtgg gcaccccaga ctacctgtcc
     2041 cccgagatcc tgcaggctgt gggcggtggg cctgggacag gcagctacgg gcccgagtgt
     2101 gactggtggg cgctgggtgt attcgcctat gaaatgttct atgggcagac gcccttctac
     2161 gcggattcca cggcggagac ctatggcaag atcgtccact acaaggagca cctctctctg
     2221 ccgctggtgg acgaaggggt ccctgaggag gctcgagact tcattcagcg gttgctgtgt
     2281 cccccggaga cacggctggg ccggggtgga gcaggcgact tccggacaca tcccttcttc
     2341 tttggcctcg actgggatgg tctccgggac agcgtgcccc cctttacacc ggatttcgaa
     2401 ggtgccaccg acacatgcaa cttcgacttg gtggaggacg ggctcactgc catggtgagc
     2461 gggggcgggg agacactgtc ggacattcgg gaaggtgcgc cgctaggggt ccacctgcct
     2521 tttgtgggct actcctactc ctgcatggcc ctcagggaca gtgaggtccc aggccccaca
     2581 cccatggaac tggaggccga gcagctgctt gagccacacg tgcaagcgcc cagcctggag
     2641 ccctcggtgt ccccacagga tgaaacagct gaagtggcag ttccagcggc tgtccctgcg
     2701 gcagaggctg aggccgaggt gacgctgcgg gagctccagg aagccctgga ggaggaggtg
     2761 ctcacccggc agagcctgag ccgggagatg gaggccatcc gcacggacaa ccagaacttc
     2821 gccagtcaac tacgcgaggc agaggctcgg aaccgggacc tagaggcaca cgtccggcag
     2881 ttgcaggagc ggatggagtt gctgcaggca gagggagcca cagctgtcac gggggtcccc
     2941 agtccccggg ccacggatcc accttcccat ctagatggcc ccccggccgt ggctgtgggc
     3001 cagtgcccgc tggtggggcc aggccccatg caccgccgcc acctgctgct ccctgccagg
     3061 gtccctaggc ctggcctatc ggaggcgctt tccctgctcc tgttcgccgt tgttctgtct
     3121 cgtgccgccg ccctgggctg cattgggttg gtggcccacg ccggccaact caccgcagtc
     3181 tggcgccgcc caggagccgc ccgcgctccc ttggacccag ctttcttgta caaagtggtt
     3241 gatatccagc acagtggcgg ccgctcgagt ctagagggcc cgcggttcga aaccagcgga
     3301 ggcggcggaa gcgggggagg aggcagcggc ggaggcggca gcgggggcgg cggaagcgct
     3361 accggtaagc ctatccctaa ccctctcctc ggtctcgatt ctacgcgtac cggtatggct
     3421 tctaacttta ctcagttcgt tctcgtcgac aatggcggaa ctggcgacgt gactgtcgcc
     3481 ccaagcaact tcgctaacgg ggtcgctgaa tggatcagct ctaactcgcg atcacaggct
     3541 tacaaagtaa cctgtagcgt tcgtcagagc tctgcgcaga atcgcaaata caccatcaaa
     3601 gtcgaggtgc ctaaagtggc aacccagact gttggtggtg aagagcttcc tgtagccgga
     3661 tggagatctt acttaaatat ggaactaacc attccaattt tcgccacgaa ttccgactgc
     3721 gagcttattg ttaaggcaat gcaaggtctc ctaaaagatg gaaacccgat tccctcggcc
     3781 atcgcggcca actccggcat ctacggaggt ggaggtggat ctcgtacggg ttagtaatga
     3841 gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     3901 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     3961 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     4021 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     4081 ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg
     4141 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     4201 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     4261 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     4321 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtac ctagaagttc
     4381 ctattccgaa gttcctattc tctagaaagt ataggaactt ccttggccaa aaagcctgaa
     4441 ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg
     4501 atgcagctct cggagggcga agaatctcgt gctttcagct tcgatgtagg agggcgtgga
     4561 tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg
     4621 cactttgcat cggccgcgct cccgattccg gaagtgcttg acattgggga attcagcgag
     4681 agcctgacct attgcatctc ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa
     4741 accgaactgc ccgctgttct gcagccggtc gcggaggcca tggatgcgat cgctgcggcc
     4801 gatcttagcc agacgagcgg gttcggccca ttcggaccgc aaggaatcgg tcaatacact
     4861 acatggcgtg atttcatatg cgcgattgct gatccccatg tgtatcactg gcaaactgtg
     4921 atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat gctttgggcc
     4981 gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa caatgtcctg
     5041 acggacaatg gccgcataac agcggtcatt gactggagcg aggcgatgtt cggggattcc
     5101 caatacgagg tcgccaacat cttcttctgg aggccgtggt tggcttgtat ggagcagcag
     5161 acgcgctact tcgagcggag gcatccggag cttgcaggat cgccgcggct ccgggcgtat
     5221 atgctccgca ttggtcttga ccaactctat cagagcttgg ttgacggcaa tttcgatgat
     5281 gcagcttggg cgcagggtcg atgcgacgca atcgtccgat ccggagccgg gactgtcggg
     5341 cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt agaagtactc
     5401 gccgatagtg gaaaccgacg ccccagcact cgtccgaggg caaaggaata gcacgtacta
     5461 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     5521 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
     5581 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     5641 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     5701 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
     5761 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     5821 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     5881 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     5941 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     6001 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     6061 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     6121 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     6181 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     6241 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     6301 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     6361 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     6421 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     6481 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     6541 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     6601 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     6661 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     6721 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     6781 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     6841 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     6901 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     6961 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     7021 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     7081 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     7141 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     7201 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     7261 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     7321 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     7381 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     7441 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     7501 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     7561 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     7621 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     7681 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     7741 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     7801 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     7861 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga
     7921 gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     7981 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt
     8041 aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc
     8101 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta
     8161 ggcttttgca aaaagctttg caaagatgga taaagtttta aacagagagg aatctttgca
     8221 gctaatggac cttctaggtc ttgaaaggag tgggaatt
//
