LOCUS       sequence-375986-        7249 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 7249)
  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 7249)
  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: PPAPDC2
FEATURES             Location/Qualifiers
     source          1..7249
                     /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"
     regulatory      1403..1412
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(2206..2230)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2355..2396
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2851..2868)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2857..3081
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3214..3233)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3365..3412
                     /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             3420..4442
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3420..4442
                     /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    4572..4693
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4609..4628)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4663..4682
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4742..4758)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4742..4758)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4755..4777)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4766..4782
                     /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(4790..4796,4797..4814,4815..4820))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4835..4856
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4973..4990)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5144..5732)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5224..5243)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5903..6694,6695..6763))
                     /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     6526..6545
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6764..6868)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6943..6962)
                     /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 gttggcacca
     1321 tgccaagtcc ccggaggagc atggagggac ggccgctggg cgtctccgct tcgagcagca
     1381 gcagcagccc cggcagccca gcccatggcg gcggtggcgg cggcagcagg tttgagttcc
     1441 agtccctgct cagcagccgc gccacggccg tggaccccac ctgcgcccgg ctccgtgcat
     1501 cggagagccc agttcaccgc cgcggctcct tccccctggc cgcggcgggc ccctcgcagt
     1561 cgcccgcgcc tccgctgccc gaggaggacc gcatggactt gaacccgtcc ttcctgggca
     1621 tcgccctgcg ctccctgctg gccatcgacc tgtggctgtc caagaagctg ggggtgtgcg
     1681 cgggagagag ctcgtcgtgg ggcagcgtgc gaccccttat gaagctgctg gagatctggg
     1741 gacacggcat cccctggctg ctgggcaccc tctactgcct gtgcaggagc gacagctggg
     1801 ccgggcgcga ggtgctgatg aacctgctct tcgccctgct gttggacctg ctgctggtgg
     1861 ccttgatcaa agggctggtc cgcaggcgcc gcccggccca caaccagatg gacatgtttg
     1921 tcactctctc ggtggacaag tactccttcc cctcgggcca tgccacaagg gccgccctga
     1981 tgtcgaggtt catcctgaac cacctggtgc tggccattcc actgagggtg ctggtggttc
     2041 tgtgggcctt cgtcttgggc ctatccaggg tcatgctggg gcggcacaat gtcaccgacg
     2101 tagcttttgg cttttttctg ggctacatgc agtacagcat cgtggactat tgctggctct
     2161 caccccataa tgctccggtc ctctttttac tgtggagtca acgattgcca actttcttgt
     2221 acaaagtggt tgatatccag cacagtggcg gccgctcgag tctagagggc ccgcggttcg
     2281 aaaccagcgg aggcggcgga agcgggggag gaggcagcgg cggaggcggc agcgggggcg
     2341 gcggaagcgc taccggtaag cctatcccta accctctcct cggtctcgat tctacgcgta
     2401 ccggtatggc ttctaacttt actcagttcg ttctcgtcga caatggcgga actggcgacg
     2461 tgactgtcgc cccaagcaac ttcgctaacg gggtcgctga atggatcagc tctaactcgc
     2521 gatcacaggc ttacaaagta acctgtagcg ttcgtcagag ctctgcgcag aatcgcaaat
     2581 acaccatcaa agtcgaggtg cctaaagtgg caacccagac tgttggtggt gaagagcttc
     2641 ctgtagccgg atggagatct tacttaaata tggaactaac cattccaatt ttcgccacga
     2701 attccgactg cgagcttatt gttaaggcaa tgcaaggtct cctaaaagat ggaaacccga
     2761 ttccctcggc catcgcggcc aactccggca tctacggagg tggaggtgga tctcgtacgg
     2821 gttagtaatg agtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat
     2881 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     2941 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     3001 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     3061 gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt
     3121 atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     3181 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     3241 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     3301 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     3361 cctagaagtt cctattccga agttcctatt ctctagaaag tataggaact tccttggcca
     3421 aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg
     3481 tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag
     3541 gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt
     3601 atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg
     3661 aattcagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag
     3721 acctgcctga aaccgaactg cccgctgttc tgcagccggt cgcggaggcc atggatgcga
     3781 tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg
     3841 gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact
     3901 ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga
     3961 tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca
     4021 acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt
     4081 tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta
     4141 tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc
     4201 tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca
     4261 atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg
     4321 ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg
     4381 tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat
     4441 agcacgtact acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     4501 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     4561 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     4621 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     4681 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
     4741 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     4801 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     4861 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     4921 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     4981 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     5041 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     5101 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     5161 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     5221 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     5281 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     5341 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     5401 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     5461 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     5521 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     5581 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     5641 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     5701 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     5761 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     5821 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     5881 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     5941 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     6001 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     6061 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     6121 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     6181 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     6241 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     6301 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     6361 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     6421 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     6481 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     6541 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     6601 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     6661 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     6721 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     6781 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     6841 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg
     6901 acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc
     6961 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc
     7021 gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct
     7081 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg
     7141 attattgact aggcttttgc aaaaagcttt gcaaagatgg ataaagtttt aaacagagag
     7201 gaatctttgc agctaatgga ccttctaggt cttgaaagga gtgggaatt
//
