LOCUS       sequence-376969-        7297 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 7297)
  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 7297)
  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: PHF6
FEATURES             Location/Qualifiers
     source          1..7297
                     /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(2254..2278)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2403..2444
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2899..2916)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2905..3129
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3262..3281)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3413..3460
                     /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             3468..4490
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3468..4490
                     /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    4620..4741
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4657..4676)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4711..4730
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4790..4806)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4790..4806)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4803..4825)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4814..4830
                     /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(4838..4844,4845..4862,4863..4868))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4883..4904
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5021..5038)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5192..5780)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5272..5291)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5951..6742,6743..6811))
                     /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     6574..6593
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6812..6916)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6991..7010)
                     /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 caagctcagt tgaacagaaa aaagggccta caagacagcg caaatgtggc ttttgtaagt
     1381 caaatagaga caaggaatgt ggacagttac taatatctga aaaccagaag gtggcagcgc
     1441 accataagtg catgctcttt tcatctgctt tggtatcatc acactctgat aatgaaagtc
     1501 ttggtggatt ttctattgaa gatgtccaaa aggaaattaa aagaggcacg aagctgatgt
     1561 gttctttgtg ccattgtcct ggagcaacaa ttggttgtga tgtgaaaaca tgtcacagga
     1621 cataccacta ccactgtgca ttgcatgata aagctcaaat acgagagaaa ccttcacaag
     1681 gaatttacat ggcctattgc cgaaaacaca agaaaactgc acataactcc gaagcagctg
     1741 atttagaaga aagttttaat gaacatgaac tggagccctc atcacctaaa agtaaaaaga
     1801 aaagtcgcaa aggaaggcca agaaaaacta attttaaagg gctgtcagaa gataccaggt
     1861 ccacatcctc ccatggaaca gatgaaatgg aaagtagttc ctatagagat aggtctccac
     1921 acagaagcag ccctagtgac accaggccta aatgtggatt ttgccatgta ggggaggaag
     1981 aaaatgaagc acgaggaaaa ctgcatatat ttaatgccaa gaaggcagct gcccattata
     2041 agtgcatgtt gttttcttct ggcacagtcc agctcacaac aacatcaaga gcagaatttg
     2101 gagactttga tattaaaact gtacttcagg agattaaacg aggaaaaaga atggtctgta
     2161 gtttttatat ttgttatgca acattacact tgatttgctg ctttaaattt agagtacatc
     2221 ccaaatttat ccagtcatca gaaaatttaa agtacccaac tttcttgtac aaagtggttg
     2281 atatccagca cagtggcggc cgctcgagtc tagagggccc gcggttcgaa accagcggag
     2341 gcggcggaag cgggggagga ggcagcggcg gaggcggcag cgggggcggc ggaagcgcta
     2401 ccggtaagcc tatccctaac cctctcctcg gtctcgattc tacgcgtacc ggtatggctt
     2461 ctaactttac tcagttcgtt ctcgtcgaca atggcggaac tggcgacgtg actgtcgccc
     2521 caagcaactt cgctaacggg gtcgctgaat ggatcagctc taactcgcga tcacaggctt
     2581 acaaagtaac ctgtagcgtt cgtcagagct ctgcgcagaa tcgcaaatac accatcaaag
     2641 tcgaggtgcc taaagtggca acccagactg ttggtggtga agagcttcct gtagccggat
     2701 ggagatctta cttaaatatg gaactaacca ttccaatttt cgccacgaat tccgactgcg
     2761 agcttattgt taaggcaatg caaggtctcc taaaagatgg aaacccgatt ccctcggcca
     2821 tcgcggccaa ctccggcatc tacggaggtg gaggtggatc tcgtacgggt tagtaatgag
     2881 tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     2941 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     3001 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     3061 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
     3121 gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc
     3181 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     3241 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     3301 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     3361 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtacc tagaagttcc
     3421 tattccgaag ttcctattct ctagaaagta taggaacttc cttggccaaa aagcctgaac
     3481 tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc tccgacctga
     3541 tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga gggcgtggat
     3601 atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat gtttatcggc
     3661 actttgcatc ggccgcgctc ccgattccgg aagtgcttga cattggggaa ttcagcgaga
     3721 gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa
     3781 ccgaactgcc cgctgttctg cagccggtcg cggaggccat ggatgcgatc gctgcggccg
     3841 atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt caatacacta
     3901 catggcgtga tttcatatgc gcgattgctg atccccatgt gtatcactgg caaactgtga
     3961 tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg ctttgggccg
     4021 aggactgccc cgaagtccgg cacctcgtgc acgcggattt cggctccaac aatgtcctga
     4081 cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc ggggattccc
     4141 aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga
     4201 cgcgctactt cgagcggagg catccggagc ttgcaggatc gccgcggctc cgggcgtata
     4261 tgctccgcat tggtcttgac caactctatc agagcttggt tgacggcaat ttcgatgatg
     4321 cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc
     4381 gtacacaaat cgcccgcaga agcgcggccg tctggaccga tggctgtgta gaagtactcg
     4441 ccgatagtgg aaaccgacgc cccagcactc gtccgagggc aaaggaatag cacgtactac
     4501 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     4561 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca
     4621 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     4681 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     4741 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     4801 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     4861 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     4921 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     4981 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     5041 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     5101 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     5161 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     5221 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     5281 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     5341 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     5401 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     5461 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     5521 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     5581 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     5641 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     5701 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     5761 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     5821 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     5881 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     5941 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     6001 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     6061 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     6121 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     6181 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     6241 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     6301 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     6361 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     6421 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     6481 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     6541 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     6601 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     6661 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     6721 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     6781 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     6841 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     6901 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag
     6961 atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     7021 ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta
     7081 agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg
     7141 ttttgcgctg cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag
     7201 gcttttgcaa aaagctttgc aaagatggat aaagttttaa acagagagga atctttgcag
     7261 ctaatggacc ttctaggtct tgaaaggagt gggaatt
//
