LOCUS       sequence-376029-        7894 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 7894)
  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 7894)
  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: SMG6
FEATURES             Location/Qualifiers
     source          1..7894
                     /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(2851..2875)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3000..3041
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3496..3513)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3502..3726
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3859..3878)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4010..4057
                     /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             4065..5087
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4065..5087
                     /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    5217..5338
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5254..5273)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5308..5327
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5387..5403)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5387..5403)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5400..5422)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5411..5427
                     /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(5435..5441,5442..5459,5460..5465))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5480..5501
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5618..5635)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5789..6377)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5869..5888)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6548..7339,7340..7408))
                     /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     7171..7190
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7409..7513)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7588..7607)
                     /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 agacattccc tgcagtggct gagaaggtcc tcaaggagtt ccaggtgtta ctgcagcaca
     1381 gcccctctcc cattggaagt acccgcatgc tgcagcttat gaccatcaat atgtttgcag
     1441 tacacaactc ccagctgaaa gactgcttct cggaggagtg ccgctctgtg atccaggaac
     1501 aagccgcagc tctgggcttg gccatgtttt ctctactggt ccgccgctgc acctgcttac
     1561 ttaaggagtc cgccaaagct cagctgtcct ctcctgagga ccaggatgac caagacgaca
     1621 tcaaggtgtc ttcctttgtc ccggacctga aggagctgct ccccagtgtc aaagtctggt
     1681 cagattggat gctcggctac ccggacacct ggaatcctcc tcccacatcc ctggatctgc
     1741 cctcgcatgt tgctgtggat gtatggtcga cgctggctga tttctgtaac atactgactg
     1801 cagtgaatca gtctgaggtg ccactgtaca aggacccgga tgatgacctc acccttctta
     1861 tcctggaaga ggatcggctt ctctcgggct ttgtcccctt gctggctgcc cctcaggacc
     1921 cctgctacgt ggagaaaacc tcggataagg ttattgcagc tgactgcaaa agggtcacag
     1981 tgctgaagta ttttctggaa gccctttgtg gacaagaaga gcctctgctg gcattcaagg
     2041 gtggaaagta tgtgtcagtg gcacccgtcc cagacaccat gggaaaggaa atgggaagcc
     2101 aagagggaac acgactggaa gatgaggagg aggatgtggt gattgaagac tttgaggaag
     2161 attcagaggc tgaaggcagc ggaggcgagg atgacatcag ggagcttcgg gccaagaagc
     2221 tggctctggc caggaagata gctgagcagc agcgtcgcca ggaaaagatc caggctgtcc
     2281 tggaggacca cagtcagatg aggcagatgg agctcgaaat cagacctttg ttcctcgtac
     2341 cagacaccaa cggcttcatt gaccacctgg ccagtctggc gcggctgctg gagagcagga
     2401 agtacatcct ggtggtgccc ctcatcgtga tcaatgagct ggacggcctg gccaaggggc
     2461 aggagacaga ccaccgggct gggggctacg cccgtgtggt acaagagaag gcccgcaagt
     2521 ccatcgagtt cctcgagcag cgattcgaga gtcgggactc ttgcctgcga gccctgacca
     2581 gccgtggcaa tgaactcgaa tccatcgcct tccgcagtga ggacatcact ggccagctgg
     2641 gtaacaacga tgatctcatc ctgtcctgct gcctccacta ctgcaaagac aaggctaagg
     2701 acttcatgcc cgccagcaaa gaggagccaa tccggctact gcgggaggtg gtgctgttga
     2761 cggatgaccg gaacctgcgt gtgaaggcgc tcacaaggaa tgttcctgta cgggacatcc
     2821 cagccttcct cacgtgggcc caggtgggct gcccaacttt cttgtacaaa gtggttgata
     2881 tccagcacag tggcggccgc tcgagtctag agggcccgcg gttcgaaacc agcggaggcg
     2941 gcggaagcgg gggaggaggc agcggcggag gcggcagcgg gggcggcgga agcgctaccg
     3001 gtaagcctat ccctaaccct ctcctcggtc tcgattctac gcgtaccggt atggcttcta
     3061 actttactca gttcgttctc gtcgacaatg gcggaactgg cgacgtgact gtcgccccaa
     3121 gcaacttcgc taacggggtc gctgaatgga tcagctctaa ctcgcgatca caggcttaca
     3181 aagtaacctg tagcgttcgt cagagctctg cgcagaatcg caaatacacc atcaaagtcg
     3241 aggtgcctaa agtggcaacc cagactgttg gtggtgaaga gcttcctgta gccggatgga
     3301 gatcttactt aaatatggaa ctaaccattc caattttcgc cacgaattcc gactgcgagc
     3361 ttattgttaa ggcaatgcaa ggtctcctaa aagatggaaa cccgattccc tcggccatcg
     3421 cggccaactc cggcatctac ggaggtggag gtggatctcg tacgggttag taatgagttt
     3481 aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     3541 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     3601 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     3661 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     3721 ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct
     3781 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     3841 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     3901 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     3961 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtacctag aagttcctat
     4021 tccgaagttc ctattctcta gaaagtatag gaacttcctt ggccaaaaag cctgaactca
     4081 ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc gacctgatgc
     4141 agctctcgga gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg
     4201 tcctgcgggt aaatagctgc gccgatggtt tctacaaaga tcgttatgtt tatcggcact
     4261 ttgcatcggc cgcgctcccg attccggaag tgcttgacat tggggaattc agcgagagcc
     4321 tgacctattg catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg
     4381 aactgcccgc tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc
     4441 ttagccagac gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa tacactacat
     4501 ggcgtgattt catatgcgcg attgctgatc cccatgtgta tcactggcaa actgtgatgg
     4561 acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg
     4621 actgccccga agtccggcac ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg
     4681 acaatggccg cataacagcg gtcattgact ggagcgaggc gatgttcggg gattcccaat
     4741 acgaggtcgc caacatcttc ttctggaggc cgtggttggc ttgtatggag cagcagacgc
     4801 gctacttcga gcggaggcat ccggagcttg caggatcgcc gcggctccgg gcgtatatgc
     4861 tccgcattgg tcttgaccaa ctctatcaga gcttggttga cggcaatttc gatgatgcag
     4921 cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg agccgggact gtcgggcgta
     4981 cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg
     5041 atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa ggaatagcac gtactacgag
     5101 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     5161 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact
     5221 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     5281 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     5341 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     5401 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     5461 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     5521 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     5581 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     5641 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     5701 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     5761 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     5821 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     5881 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     5941 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     6001 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     6061 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     6121 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     6181 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     6241 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     6301 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     6361 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     6421 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     6481 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     6541 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     6601 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     6661 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     6721 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     6781 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     6841 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     6901 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     6961 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     7021 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     7081 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     7141 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     7201 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     7261 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     7321 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     7381 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     7441 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     7501 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc
     7561 tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     7621 gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc
     7681 tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt
     7741 tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat tgactaggct
     7801 tttgcaaaaa gctttgcaaa gatggataaa gttttaaaca gagaggaatc tttgcagcta
     7861 atggaccttc taggtcttga aaggagtggg aatt
//
