LOCUS       sequence-375990-        7351 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 7351)
  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 7351)
  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: PPP1CA
FEATURES             Location/Qualifiers
     source          1..7351
                     /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(2308..2332)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2457..2498
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2953..2970)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2959..3183
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3316..3335)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3467..3514
                     /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             3522..4544
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3522..4544
                     /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    4674..4795
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4711..4730)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4765..4784
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4844..4860)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4844..4860)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4857..4879)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4868..4884
                     /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(4892..4898,4899..4916,4917..4922))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4937..4958
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5075..5092)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5246..5834)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5326..5345)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6005..6796,6797..6865))
                     /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     6628..6647
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6866..6970)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7045..7064)
                     /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 gttggcatgt
     1321 ccgacagcga gaagctcaac ctggactcga tcatcgggcg cctgctggaa gtgcagggct
     1381 cgcggcctgg caagaatgta cagctgacag agaacgagat ccgcggtctg tgcctgaaat
     1441 cccgggagat ttttctgagc cagcccattc ttctggagct ggaggcaccc ctcaagatct
     1501 gcggtgacat acacggccag tactacgacc ttctgcgact atttgagtat ggcggtttcc
     1561 ctcccgagag caactacctc tttctggggg actatgtgga caggggcaag cagtccttgg
     1621 agaccatctg cctgctgctg gcctataaga tcaagtaccc cgagaacttc ttcctgctcc
     1681 gtgggaacca cgagtgtgcc agcatcaacc gcatctatgg tttctacgat gagtgcaaga
     1741 gacgctacaa catcaaactg tggaaaacct tcactgactg cttcaactgc ctgcccatcg
     1801 cggccatagt ggacgaaaag atcttctgct gccacggagg cctgtccccg gacctgcagt
     1861 ctatggagca gattcggcgg atcatgcggc ccacagatgt gcctgaccag ggcctgctgt
     1921 gtgacctgct gtggtctgac cctgacaagg acgtgcaggg ctggggcgag aacgaccgtg
     1981 gcgtctcttt tacctttgga gccgaggtgg tggccaagtt cctccacaag cacgacttgg
     2041 acctcatctg ccgagcacac caggtggtag aagacggcta cgagttcttt gccaagcggc
     2101 agctggtgac acttttctca gctcccaact actgtggcga gtttgacaat gctggcgcca
     2161 tgatgagtgt ggacgagacc ctcatgtgct ctttccagat cctcaagccc gccgacaaga
     2221 acaaggggaa gtacgggcag ttcagtggcc tgaaccctgg aggccgaccc atcaccccac
     2281 cccgcaattc cgccaaagcc aagaaatacc caactttctt gtacaaagtg gttgatatcc
     2341 agcacagtgg cggccgctcg agtctagagg gcccgcggtt cgaaaccagc ggaggcggcg
     2401 gaagcggggg aggaggcagc ggcggaggcg gcagcggggg cggcggaagc gctaccggta
     2461 agcctatccc taaccctctc ctcggtctcg attctacgcg taccggtatg gcttctaact
     2521 ttactcagtt cgttctcgtc gacaatggcg gaactggcga cgtgactgtc gccccaagca
     2581 acttcgctaa cggggtcgct gaatggatca gctctaactc gcgatcacag gcttacaaag
     2641 taacctgtag cgttcgtcag agctctgcgc agaatcgcaa atacaccatc aaagtcgagg
     2701 tgcctaaagt ggcaacccag actgttggtg gtgaagagct tcctgtagcc ggatggagat
     2761 cttacttaaa tatggaacta accattccaa ttttcgccac gaattccgac tgcgagctta
     2821 ttgttaaggc aatgcaaggt ctcctaaaag atggaaaccc gattccctcg gccatcgcgg
     2881 ccaactccgg catctacgga ggtggaggtg gatctcgtac gggttagtaa tgagtttaaa
     2941 cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     3001 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     3061 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     3121 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     3181 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
     3241 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     3301 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     3361 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     3421 acctcgaccc caaaaaactt gattagggtg atggttcacg tacctagaag ttcctattcc
     3481 gaagttccta ttctctagaa agtataggaa cttccttggc caaaaagcct gaactcaccg
     3541 cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc
     3601 tctcggaggg cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc
     3661 tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg
     3721 catcggccgc gctcccgatt ccggaagtgc ttgacattgg ggaattcagc gagagcctga
     3781 cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac
     3841 tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta
     3901 gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc
     3961 gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg
     4021 acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact
     4081 gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca
     4141 atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg
     4201 aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct
     4261 acttcgagcg gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc
     4321 gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt
     4381 gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac
     4441 aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata
     4501 gtggaaaccg acgccccagc actcgtccga gggcaaagga atagcacgta ctacgagatt
     4561 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     4621 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     4681 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     4741 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     4801 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     4861 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     4921 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     4981 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     5041 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     5101 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     5161 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     5221 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     5281 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     5341 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     5401 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     5461 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     5521 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     5581 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     5641 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     5701 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     5761 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     5821 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     5881 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     5941 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     6001 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     6061 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     6121 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     6181 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     6241 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     6301 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     6361 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     6421 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     6481 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     6541 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     6601 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     6661 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     6721 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     6781 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     6841 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     6901 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     6961 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc
     7021 cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     7081 tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac
     7141 aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc
     7201 gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctaggctttt
     7261 gcaaaaagct ttgcaaagat ggataaagtt ttaaacagag aggaatcttt gcagctaatg
     7321 gaccttctag gtcttgaaag gagtgggaat t
//
