LOCUS       sequence-376760-        7366 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 7366)
  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 7366)
  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: GAPDH
FEATURES             Location/Qualifiers
     source          1..7366
                     /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(2323..2347)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2472..2513
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2968..2985)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2974..3198
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3331..3350)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3482..3529
                     /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             3537..4559
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3537..4559
                     /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    4689..4810
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4726..4745)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4780..4799
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4859..4875)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4859..4875)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4872..4894)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4883..4899
                     /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(4907..4913,4914..4931,4932..4937))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4952..4973
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5090..5107)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5261..5849)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5341..5360)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6020..6811,6812..6880))
                     /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     6643..6662
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6881..6985)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7060..7079)
                     /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 ggaaggtgaa ggtcggagtc aacggatttg gtcgtattgg gcgcctggtc accagggctg
     1381 cttttaactc tggtaaagtg gatattgttg ccatcaatga ccccttcatt gacctcaact
     1441 acatggttta catgttccaa tatgattcca cccatggcaa attccatggc accgtcaagg
     1501 ctgagaacgg gaagcttgtc atcaatggaa atcccatcac catcttccag gagcgagatc
     1561 cctccaaaat caagtggggc gatgctggcg ctgagtacgt cgtggagtcc actggcgtct
     1621 tcaccaccat ggagaaggct ggggctcatt tgcagggggg agccaaaagg gtcatcatct
     1681 ctgccccctc tgctgatgcc cccatgttcg tcatgggtgt gaaccatgag aagtatgaca
     1741 acagcctcaa gatcatcagc aatgcctcct gcaccaccaa ctgcttagca cccctggcca
     1801 aggtcatcca tgacaacttt ggtatcgtgg aaggactcat gaccacagtc catgccatca
     1861 ctgccaccca gaagactgtg gatggcccct ccgggaaact gtggcgtgat ggccgcgggg
     1921 ctctccagaa catcatccct gcctctactg gcgctgccaa ggctgtgggc aaggtcatcc
     1981 ctgagctgaa cgggaagctc actggcatgg ccttccgtgt ccccactgcc aacgtgtcag
     2041 tggtggacct gacctgccgt ctagaaaaac ctgccaaata tgatgacatc aagaaggtgg
     2101 tgaagcaggc gtcggagggc cccctcaagg gcatcctggg ctacactgag caccaggtgg
     2161 tctcctctga cttcaacagc gacacccact cctccacctt tgacgctggg gctggcattg
     2221 ccctcaacga ccactttgtc aagctcattt cctggtatga caacgaattt ggctacagca
     2281 acagggtggt ggacctcatg gcccacatgg cctccaagga gtacccaact ttcttgtaca
     2341 aagtggttga tatccagcac agtggcggcc gctcgagtct agagggcccg cggttcgaaa
     2401 ccagcggagg cggcggaagc gggggaggag gcagcggcgg aggcggcagc gggggcggcg
     2461 gaagcgctac cggtaagcct atccctaacc ctctcctcgg tctcgattct acgcgtaccg
     2521 gtatggcttc taactttact cagttcgttc tcgtcgacaa tggcggaact ggcgacgtga
     2581 ctgtcgcccc aagcaacttc gctaacgggg tcgctgaatg gatcagctct aactcgcgat
     2641 cacaggctta caaagtaacc tgtagcgttc gtcagagctc tgcgcagaat cgcaaataca
     2701 ccatcaaagt cgaggtgcct aaagtggcaa cccagactgt tggtggtgaa gagcttcctg
     2761 tagccggatg gagatcttac ttaaatatgg aactaaccat tccaattttc gccacgaatt
     2821 ccgactgcga gcttattgtt aaggcaatgc aaggtctcct aaaagatgga aacccgattc
     2881 cctcggccat cgcggccaac tccggcatct acggaggtgg aggtggatct cgtacgggtt
     2941 agtaatgagt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     3001 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     3061 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     3121 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     3181 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     3241 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     3301 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     3361 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     3421 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtacct
     3481 agaagttcct attccgaagt tcctattctc tagaaagtat aggaacttcc ttggccaaaa
     3541 agcctgaact caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct
     3601 ccgacctgat gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag
     3661 ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg
     3721 tttatcggca ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggaat
     3781 tcagcgagag cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc
     3841 tgcctgaaac cgaactgccc gctgttctgc agccggtcgc ggaggccatg gatgcgatcg
     3901 ctgcggccga tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc
     3961 aatacactac atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc
     4021 aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc
     4081 tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca
     4141 atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg
     4201 gggattccca atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg
     4261 agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc
     4321 gggcgtatat gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt
     4381 tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga
     4441 ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag
     4501 aagtactcgc cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaatagc
     4561 acgtactacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     4621 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     4681 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     4741 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     4801 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     4861 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     4921 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     4981 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     5041 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     5101 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     5161 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     5221 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     5281 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     5341 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     5401 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     5461 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     5521 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     5581 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     5641 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     5701 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     5761 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     5821 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     5881 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     5941 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     6001 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     6061 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     6121 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     6181 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     6241 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     6301 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     6361 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     6421 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     6481 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     6541 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     6601 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     6661 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     6721 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     6781 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     6841 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     6901 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     6961 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg
     7021 gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc
     7081 atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag
     7141 caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag
     7201 ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt
     7261 attgactagg cttttgcaaa aagctttgca aagatggata aagttttaaa cagagaggaa
     7321 tctttgcagc taatggacct tctaggtctt gaaaggagtg ggaatt
//
