LOCUS       sequence-376935-        8110 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 8110)
  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 8110)
  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: METTL3
FEATURES             Location/Qualifiers
     source          1..8110
                     /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(3067..3091)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3216..3257
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3712..3729)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3718..3942
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4075..4094)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4226..4273
                     /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             4281..5303
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4281..5303
                     /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    5433..5554
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5470..5489)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5524..5543
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5603..5619)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5603..5619)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5616..5638)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5627..5643
                     /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(5651..5657,5658..5675,5676..5681))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5696..5717
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5834..5851)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6005..6593)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6085..6104)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6764..7555,7556..7624))
                     /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     7387..7406
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7625..7729)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7804..7823)
                     /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 gcaggctcca
     1321 ccatgtcgga cacgtggagc tctatccagg cccacaagaa gcagctggac tctctgcggg
     1381 agaggctgca gcggaggcgg aagcaggact cggggcactt ggatctacgg aatccagagg
     1441 cagcattgtc tccaaccttc cgtagtgaca gcccagtgcc tactgcaccc acctctggtg
     1501 gccctaagcc cagcacagct tcagcagttc ctgaattagc tacagatcct gagttagaga
     1561 agaagttgct acaccacctc tctgatctgg ccttaacatt gcccactgat gctgtgtcca
     1621 tctgtcttgc catctccacg ccagatgctc ctgccactca agatggggta gaaagcctcc
     1681 tgcagaagtt tgcagctcag gagttgattg aggtaaagcg aggtctccta caagatgatg
     1741 cacatcctac tcttgtaacc tatgctgacc attccaagct ctctgccatg atgggtgctg
     1801 tggcagaaaa gaagggccct ggggaggtag cagggactgt cacagggcag aagcggcgtg
     1861 cagaacagga ctcgactaca gtagctgcct ttgccagttc gttagtctct ggtctgaact
     1921 cttcagcatc ggaaccagca aaggagccag ccaagaaatc aaggaaacat gctgcctcag
     1981 atgttgatct ggagatagag agccttctga accaacagtc cactaaggaa caacagagca
     2041 agaaggtcag tcaggagatc ctagagctat taaatactac aacagccaag gaacaatcca
     2101 ttgttgaaaa atttcgctct cgaggtcggg cccaagtgca agaattctgt gactatggaa
     2161 ccaaggagga gtgcatgaaa gccagtgatg ctgatcgacc ctgtcgcaag ctgcacttca
     2221 gacgaattat caataaacac actgatgagt ctttaggtga ctgctctttc cttaatacat
     2281 gtttccacat ggatacctgc aagtatgttc aatatgaaat tgatgcttgc atggattctg
     2341 aggcccctgg cagcaaagac cacacgccaa gccaggagct tgctcttaca cagagtgtcg
     2401 gaggtgattc cagtgcagac cgactcttcc cacctcagtg gatctgttgt gatatccgct
     2461 acctggacgt cagtatcttg ggcaagtttg cagttgtgat ggctgaccca ccctgggata
     2521 ttcacatgga actgccctat gggaccctga cagatgatga gatgcgcagg ctcaacatac
     2581 ccgtactaca ggatgatggc tttctcttcc tctgggtcac aggcagggcc atggagttgg
     2641 ggagagaatg tctaaacctc tgggggtatg aacgggtaga tgaaattatt tgggtgaaga
     2701 caaatcaact gcaacgcatc attcggacag gccgtacagg tcactggttg aaccatggga
     2761 aggaacactg cttggttggt gtcaaaggaa atccccaagg cttcaaccag ggtctggatt
     2821 gtgatgtgat cgtagctgag gttcgttcca ccagtcataa accagatgaa atctatggca
     2881 tgattgaaag actatctcct ggcactcgca agattgagtt atttggacga ccacacaatg
     2941 tgcaacccaa ctggatcacc cttggaaacc aactggatgg gatccaccta ctagacccag
     3001 atgtggttgc acggttcaag caaaagtacc cagatggtat catctctaaa cctaagaatt
     3061 tattggaccc agctttcttg tacaaagtgg ttgatatcca gcacagtggc ggccgctcga
     3121 gtctagaggg cccgcggttc gaaaccagcg gaggcggcgg aagcggggga ggaggcagcg
     3181 gcggaggcgg cagcgggggc ggcggaagcg ctaccggtaa gcctatccct aaccctctcc
     3241 tcggtctcga ttctacgcgt accggtatgg cttctaactt tactcagttc gttctcgtcg
     3301 acaatggcgg aactggcgac gtgactgtcg ccccaagcaa cttcgctaac ggggtcgctg
     3361 aatggatcag ctctaactcg cgatcacagg cttacaaagt aacctgtagc gttcgtcaga
     3421 gctctgcgca gaatcgcaaa tacaccatca aagtcgaggt gcctaaagtg gcaacccaga
     3481 ctgttggtgg tgaagagctt cctgtagccg gatggagatc ttacttaaat atggaactaa
     3541 ccattccaat tttcgccacg aattccgact gcgagcttat tgttaaggca atgcaaggtc
     3601 tcctaaaaga tggaaacccg attccctcgg ccatcgcggc caactccggc atctacggag
     3661 gtggaggtgg atctcgtacg ggttagtaat gagtttaaac ccgctgatca gcctcgactg
     3721 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     3781 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     3841 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     3901 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     3961 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
     4021 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     4081 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     4141 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     4201 attagggtga tggttcacgt acctagaagt tcctattccg aagttcctat tctctagaaa
     4261 gtataggaac ttccttggcc aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc
     4321 tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc gaagaatctc
     4381 gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg
     4441 atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg ctcccgattc
     4501 cggaagtgct tgacattggg gaattcagcg agagcctgac ctattgcatc tcccgccgtg
     4561 cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg
     4621 tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc gggttcggcc
     4681 cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata tgcgcgattg
     4741 ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt gcgtccgtcg
     4801 cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc cggcacctcg
     4861 tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata acagcggtca
     4921 ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac atcttcttct
     4981 ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg aggcatccgg
     5041 agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt gaccaactct
     5101 atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg
     5161 caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg
     5221 ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga cgccccagca
     5281 ctcgtccgag ggcaaaggaa tagcacgtac tacgagattt cgattccacc gccgccttct
     5341 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     5401 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     5461 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     5521 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     5581 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     5641 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     5701 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     5761 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     5821 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     5881 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     5941 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     6001 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     6061 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     6121 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     6181 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     6241 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     6301 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     6361 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     6421 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     6481 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     6541 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     6601 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     6661 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     6721 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     6781 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     6841 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     6901 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     6961 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     7021 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     7081 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     7141 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     7201 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     7261 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     7321 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     7381 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     7441 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     7501 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     7561 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     7621 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     7681 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     7741 gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct
     7801 cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
     7861 ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga
     7921 caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
     7981 cagatatacg cgttgacatt gattattgac taggcttttg caaaaagctt tgcaaagatg
     8041 gataaagttt taaacagaga ggaatctttg cagctaatgg accttctagg tcttgaaagg
     8101 agtgggaatt
//
