LOCUS       sequence-377084-        7144 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 7144)
  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 7144)
  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: THOC4
FEATURES             Location/Qualifiers
     source          1..7144
                     /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(2101..2125)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2250..2291
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2746..2763)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2752..2976
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3109..3128)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3260..3307
                     /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             3315..4337
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3315..4337
                     /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    4467..4588
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4504..4523)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4558..4577
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4637..4653)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4637..4653)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4650..4672)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4661..4677
                     /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(4685..4691,4692..4709,4710..4715))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4730..4751
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4868..4885)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5039..5627)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5119..5138)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5798..6589,6590..6658))
                     /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     6421..6440
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6659..6763)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6838..6857)
                     /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 ccatggccga caaaatggac atgtctctgg acgacatcat taaactgaac cggagccagc
     1381 gaggcggccg gggcgggggc cggggccgcg gccgggccgg ctcccagggc ggccgcggcg
     1441 gtggggcgca ggccgccgcg cgagtgaatc gaggcggcgg gcccatccgg aaccggccgg
     1501 ccatcgcccg cggcgcggcc ggcggaggcg gcaggaaccg accggcgccc tacagcaggc
     1561 caaaacaact tcccgacaag tggcagcacg atcttttcga cagtggcttc ggcggtggtg
     1621 ccggcgtgga gacaggtggg aaactgctgg tgtccaatct ggattttgga gtctcagacg
     1681 ccgatattca ggaactcttt gctgaatttg gaacgctgaa gaaggcggct gtgcactatg
     1741 atcgctctgg tcgcagctta ggaacagcag acgtgcactt tgagcggaag gcagatgccc
     1801 tgaaggccat gaagcagtac aacggcgtcc ctctggatgg ccgccccatg aacattcagc
     1861 ttgtcacgtc acagattgac gcacagcgga ggcctgcaca gagcgtaaac agaggtggca
     1921 tgactagaaa ccgtggcgct ggaggttttg gtggtggtgg aggcacccgg agaggcaccc
     1981 gcggaggcgc ccgtggaaga ggcagaggtg ccggcaggaa ttcaaagcag cagctttcgg
     2041 cagaggagct ggatgcccag ctggacgcct ataatgcgag aatggacacc agttggatcc
     2101 acccagcttt cttgtacaaa gtggttgata tccagcacag tggcggccgc tcgagtctag
     2161 agggcccgcg gttcgaaacc agcggaggcg gcggaagcgg gggaggaggc agcggcggag
     2221 gcggcagcgg gggcggcgga agcgctaccg gtaagcctat ccctaaccct ctcctcggtc
     2281 tcgattctac gcgtaccggt atggcttcta actttactca gttcgttctc gtcgacaatg
     2341 gcggaactgg cgacgtgact gtcgccccaa gcaacttcgc taacggggtc gctgaatgga
     2401 tcagctctaa ctcgcgatca caggcttaca aagtaacctg tagcgttcgt cagagctctg
     2461 cgcagaatcg caaatacacc atcaaagtcg aggtgcctaa agtggcaacc cagactgttg
     2521 gtggtgaaga gcttcctgta gccggatgga gatcttactt aaatatggaa ctaaccattc
     2581 caattttcgc cacgaattcc gactgcgagc ttattgttaa ggcaatgcaa ggtctcctaa
     2641 aagatggaaa cccgattccc tcggccatcg cggccaactc cggcatctac ggaggtggag
     2701 gtggatctcg tacgggttag taatgagttt aaacccgctg atcagcctcg actgtgcctt
     2761 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     2821 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     2881 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     2941 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     3001 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     3061 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     3121 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     3181 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     3241 gtgatggttc acgtacctag aagttcctat tccgaagttc ctattctcta gaaagtatag
     3301 gaacttcctt ggccaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg
     3361 aaaagttcga cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt
     3421 tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt
     3481 tctacaaaga tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag
     3541 tgcttgacat tggggaattc agcgagagcc tgacctattg catctcccgc cgtgcacagg
     3601 gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg
     3661 aggccatgga tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg
     3721 gaccgcaagg aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc
     3781 cccatgtgta tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg
     3841 ctctcgatga gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg
     3901 cggatttcgg ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact
     3961 ggagcgaggc gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc
     4021 cgtggttggc ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg
     4081 caggatcgcc gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga
     4141 gcttggttga cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg
     4201 tccgatccgg agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct
     4261 ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc
     4321 cgagggcaaa ggaatagcac gtactacgag atttcgattc caccgccgcc ttctatgaaa
     4381 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     4441 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     4501 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     4561 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     4621 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     4681 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     4741 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     4801 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     4861 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     4921 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     4981 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     5041 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     5101 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     5161 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     5221 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     5281 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     5341 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     5401 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     5461 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     5521 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     5581 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     5641 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     5701 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     5761 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     5821 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     5881 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     5941 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     6001 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     6061 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     6121 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     6181 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     6241 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     6301 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     6361 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     6421 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     6481 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     6541 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     6601 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     6661 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     6721 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     6781 tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac
     6841 aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt
     6901 cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg
     6961 catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata
     7021 tacgcgttga cattgattat tgactaggct tttgcaaaaa gctttgcaaa gatggataaa
     7081 gttttaaaca gagaggaatc tttgcagcta atggaccttc taggtcttga aaggagtggg
     7141 aatt
//
