LOCUS       sequence-376748-        6826 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 6826)
  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 6826)
  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: RY1
FEATURES             Location/Qualifiers
     source          1..6826
                     /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(1783..1807)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1932..1973
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2428..2445)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2434..2658
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2791..2810)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2942..2989
                     /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             2997..4019
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2997..4019
                     /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    4149..4270
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4186..4205)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4240..4259
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4319..4335)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4319..4335)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4332..4354)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4343..4359
                     /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(4367..4373,4374..4391,4392..4397))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4412..4433
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4550..4567)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4721..5309)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4801..4820)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5480..6271,6272..6340))
                     /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     6103..6122
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6341..6445)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6520..6539)
                     /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 gtcgcagtcg cagccgctct ccacggaggg aacgtaggcg ttcccggtcc acatcccggg
     1381 agagagaacg caggcgccga gaaaggtcca ggtctcggga gagagatcgg agaaggagcc
     1441 gctcgcgatc cccgcaccga agacgctccc gatctccaag acgacataga tccacatctc
     1501 cttccccttc tcgactgaaa gaaagaagag atgaggaaaa gaaagaaaca aaagaaacaa
     1561 agagcaaaga acggcagatt actgaggaag acttagaggg caaaacagag gaagaaatag
     1621 aaatgatgaa gttaatggga tttgcctcct ttgactccac aaaaggtaag aaggtggatg
     1681 gctctgtaaa tgcctatgcc ataaatgtct ctcagaagag gaagtacagg cagtacatga
     1741 atcgaaaagg tggattcaac agacctttgg atttcattgc atgcccaact ttcttgtaca
     1801 aagtggttga tatccagcac agtggcggcc gctcgagtct agagggcccg cggttcgaaa
     1861 ccagcggagg cggcggaagc gggggaggag gcagcggcgg aggcggcagc gggggcggcg
     1921 gaagcgctac cggtaagcct atccctaacc ctctcctcgg tctcgattct acgcgtaccg
     1981 gtatggcttc taactttact cagttcgttc tcgtcgacaa tggcggaact ggcgacgtga
     2041 ctgtcgcccc aagcaacttc gctaacgggg tcgctgaatg gatcagctct aactcgcgat
     2101 cacaggctta caaagtaacc tgtagcgttc gtcagagctc tgcgcagaat cgcaaataca
     2161 ccatcaaagt cgaggtgcct aaagtggcaa cccagactgt tggtggtgaa gagcttcctg
     2221 tagccggatg gagatcttac ttaaatatgg aactaaccat tccaattttc gccacgaatt
     2281 ccgactgcga gcttattgtt aaggcaatgc aaggtctcct aaaagatgga aacccgattc
     2341 cctcggccat cgcggccaac tccggcatct acggaggtgg aggtggatct cgtacgggtt
     2401 agtaatgagt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     2461 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     2521 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     2581 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     2641 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     2701 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     2761 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     2821 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     2881 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtacct
     2941 agaagttcct attccgaagt tcctattctc tagaaagtat aggaacttcc ttggccaaaa
     3001 agcctgaact caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct
     3061 ccgacctgat gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag
     3121 ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg
     3181 tttatcggca ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggaat
     3241 tcagcgagag cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc
     3301 tgcctgaaac cgaactgccc gctgttctgc agccggtcgc ggaggccatg gatgcgatcg
     3361 ctgcggccga tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc
     3421 aatacactac atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc
     3481 aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc
     3541 tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca
     3601 atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg
     3661 gggattccca atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg
     3721 agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc
     3781 gggcgtatat gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt
     3841 tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga
     3901 ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag
     3961 aagtactcgc cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaatagc
     4021 acgtactacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     4081 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     4141 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     4201 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     4261 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     4321 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     4381 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     4441 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4501 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     4561 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4621 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4681 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4741 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4801 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4861 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     4921 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4981 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     5041 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     5101 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     5161 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     5221 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     5281 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     5341 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     5401 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     5461 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     5521 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     5581 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     5641 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     5701 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5761 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     5821 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     5881 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     5941 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     6001 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     6061 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     6121 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     6181 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     6241 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     6301 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     6361 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     6421 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg
     6481 gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc
     6541 atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag
     6601 caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag
     6661 ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt
     6721 attgactagg cttttgcaaa aagctttgca aagatggata aagttttaaa cagagaggaa
     6781 tctttgcagc taatggacct tctaggtctt gaaaggagtg ggaatt
//
