LOCUS       sequence-376712-        7636 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 7636)
  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 7636)
  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: CLP1
FEATURES             Location/Qualifiers
     source          1..7636
                     /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(2593..2617)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2742..2783
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3238..3255)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3244..3468
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3601..3620)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3752..3799
                     /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             3807..4829
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3807..4829
                     /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    4959..5080
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4996..5015)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5050..5069
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5129..5145)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5129..5145)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5142..5164)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5153..5169
                     /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(5177..5183,5184..5201,5202..5207))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5222..5243
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5360..5377)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5531..6119)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5611..5630)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6290..7081,7082..7150))
                     /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     6913..6932
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7151..7255)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7330..7349)
                     /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 gagaagaggc taatgatgac aagaagccaa ccactaaatt tgaactagag cgagaaacag
     1381 aacttcgctt tgaggtggag gcatctcagt cagttcagtt ggagttgttg actggcatgg
     1441 cagagatctt tggcacagag ctgacccgaa acaagaaatt cacctttgat gctggtgcca
     1501 aggtggctgt tttcacttgg catggctgtt ctgtgcaact gagcggccgc actgaggtgg
     1561 cttatgtctc caaggacact cctatgttgc tttacctcaa cactcacaca gccttggaac
     1621 agatgcggag gcaagcggaa aaggaagaag agcgaggtcc ccgagtgatg gtagtgggcc
     1681 ccactgatgt gggcaagtct acagtgtgtc gccttctgct caactacgca gtgcgtttgg
     1741 gccgccgtcc cacttatgtg gagctggatg tgggccaggg ttctgtgtcc atccctggta
     1801 ccatgggggc cctctacatc gagcggcctg cagatgtcga agagggtttc tctatccagg
     1861 cccctctggt gtatcatttt ggttccacca ctcctggcac taacatcaag ctttataata
     1921 agattacatc tcgtttagca gatgtgttca accaaaggtg tgaggtgaac cgaagggcat
     1981 ctgtgagtgg ctgtgtcatt aacacctgtg gctgggtcaa gggctctggt taccaggctc
     2041 tggtgcatgc agcctcagct tttgaggtgg atgtcgttgt tgttctggat caagaacgac
     2101 tgtacaatga actgaaacgg gacctccccc actttgtacg cactgtgctg ctccctaaat
     2161 ctgggggtgt ggtggagcgc tccaaggact tccggcggga atgtagggat gagcgtatcc
     2221 gtgagtattt ttatggattc cgaggctgtt tctatcccca tgccttcaat gtcaaatttt
     2281 cagatgtgaa aatctacaaa gttggggcac ccaccatccc agactcctgt ttacctttgg
     2341 gcatgtctca agaggataat cagctcaagc tagtacctgt cactcctggg cgagatatgg
     2401 tgcaccacct actgagtgtt agcactgccg agggtacaga ggagaacctg tccgagacaa
     2461 gtgtagctgg cttcattgtg gtgaccagtg tggacctgga gcatcaggtg tttactgttc
     2521 tgtctccagc ccctcgccca ctgcctaaga acttccttct catcatggat atccggttca
     2581 tggatctgaa gtacccaact ttcttgtaca aagtggttga tatccagcac agtggcggcc
     2641 gctcgagtct agagggcccg cggttcgaaa ccagcggagg cggcggaagc gggggaggag
     2701 gcagcggcgg aggcggcagc gggggcggcg gaagcgctac cggtaagcct atccctaacc
     2761 ctctcctcgg tctcgattct acgcgtaccg gtatggcttc taactttact cagttcgttc
     2821 tcgtcgacaa tggcggaact ggcgacgtga ctgtcgcccc aagcaacttc gctaacgggg
     2881 tcgctgaatg gatcagctct aactcgcgat cacaggctta caaagtaacc tgtagcgttc
     2941 gtcagagctc tgcgcagaat cgcaaataca ccatcaaagt cgaggtgcct aaagtggcaa
     3001 cccagactgt tggtggtgaa gagcttcctg tagccggatg gagatcttac ttaaatatgg
     3061 aactaaccat tccaattttc gccacgaatt ccgactgcga gcttattgtt aaggcaatgc
     3121 aaggtctcct aaaagatgga aacccgattc cctcggccat cgcggccaac tccggcatct
     3181 acggaggtgg aggtggatct cgtacgggtt agtaatgagt ttaaacccgc tgatcagcct
     3241 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     3301 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     3361 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     3421 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg
     3481 aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg
     3541 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     3601 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     3661 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     3721 aacttgatta gggtgatggt tcacgtacct agaagttcct attccgaagt tcctattctc
     3781 tagaaagtat aggaacttcc ttggccaaaa agcctgaact caccgcgacg tctgtcgaga
     3841 agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag
     3901 aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct
     3961 gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc
     4021 cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc
     4081 gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc
     4141 agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt
     4201 tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg
     4261 cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt
     4321 ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc
     4381 acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag
     4441 cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct
     4501 tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc
     4561 atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc
     4621 aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat
     4681 gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa
     4741 gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc
     4801 ccagcactcg tccgagggca aaggaatagc acgtactacg agatttcgat tccaccgccg
     4861 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     4921 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     4981 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     5041 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     5101 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     5161 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     5221 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5281 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5341 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     5401 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     5461 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5521 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     5581 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     5641 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     5701 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     5761 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     5821 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     5881 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     5941 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     6001 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     6061 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     6121 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     6181 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     6241 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6301 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6361 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6421 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     6481 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     6541 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     6601 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     6661 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     6721 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     6781 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     6841 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     6901 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     6961 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     7021 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     7081 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     7141 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     7201 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     7261 ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg
     7321 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg
     7381 tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt
     7441 gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt
     7501 acgggccaga tatacgcgtt gacattgatt attgactagg cttttgcaaa aagctttgca
     7561 aagatggata aagttttaaa cagagaggaa tctttgcagc taatggacct tctaggtctt
     7621 gaaaggagtg ggaatt
//
