LOCUS       sequence-377214-        7762 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 7762)
  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 7762)
  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: SF3A2
FEATURES             Location/Qualifiers
     source          1..7762
                     /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(2719..2743)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2868..2909
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3364..3381)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3370..3594
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3727..3746)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3878..3925
                     /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             3933..4955
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3933..4955
                     /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    5085..5206
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5122..5141)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5176..5195
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5255..5271)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5255..5271)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5268..5290)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5279..5295
                     /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(5303..5309,5310..5327,5328..5333))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5348..5369
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5486..5503)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5657..6245)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5737..5756)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6416..7207,7208..7276))
                     /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     7039..7058
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7277..7381)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7456..7475)
                     /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 ccatggactt ccagcatcgc cccgggggca agaccgggag cgggggcgtg gcctcctcct
     1381 ccgagagcaa ccgtgaccgc agggagcgcc tccggcagct ggccctggag accatcgaca
     1441 tcaacaagga cccgtacttc atgaagaacc acctgggctc ctatgaatgc aaactctgcc
     1501 tgacacttca caacaatgag gggagctacc tggcacatac gcaggggaag aagcaccaga
     1561 ccaacctggc ccggcgagca gccaaggagg ccaaggaggc ccctgcccag cccgcgcctg
     1621 agaaggtcaa ggtggaggtg aagaagtttg tgaagatcgg ccgcccgggc tacaaagtga
     1681 ccaagcagag agactcggag atgggccagc agagcctcct cttccagatt gactaccctg
     1741 agatcgccga gggcatcatg ccacgtcacc gcttcatgtc tgcgtacgag cagaggatcg
     1801 agcctccgga ccggcgctgg cagtacctgc tcatggccgc cgagccctac gagaccattg
     1861 ccttcaaggt gccgagcaga gagatcgaca aggcggaggg caagttctgg acacactgga
     1921 accgggagac caagcagttc ttcctccagt tccactttaa gatggagaag cccccggctc
     1981 cacccagcct ccctgctggc ccccctgggg tgaagcggcc tccacccccg ctgatgaacg
     2041 gtctgccccc tcggccaccg ctgcctgagt ctttgccacc gcccccgcca ggaggcctgc
     2101 ctctgccacc catgcccccc acagggcctg cgccctcagg gcccccggga ccaccccagc
     2161 tacccccgcc agctccaggg gtccaccccc cggccccagt ggtgcatccc cctgcatctg
     2221 gggtccatcc cccagctcct ggcgtccacc ccccagctcc tggcgtccat cccccagccc
     2281 ctggggtcca cccaccaacc tctggggtcc accccccagc tcctggagtc caccctccag
     2341 cccccggggt tcacccacca gcccccggag tccacccacc agcccctggg gttcacccac
     2401 cagccccagg ggtccatcct cccccatcag cgggggttca cccccaggcc ccgggggtgc
     2461 acccagcagc ccccgccgtt caccctcagg ccccaggggt gcacccacca gccccaggga
     2521 tgcaccctca ggccccgggg gtccaccccc aacctcccgg ggtccatccg tcggctcctg
     2581 gggtccaccc tcagcctccg ggagttcacc cctcaaatcc tggggtgcac cccccaactc
     2641 ccatgccccc aatgctgagg cccccacttc cctccgaagg cccagggaac atacctcccc
     2701 ctcccccaac caacttggac ccagctttct tgtacaaagt ggttgatatc cagcacagtg
     2761 gcggccgctc gagtctagag ggcccgcggt tcgaaaccag cggaggcggc ggaagcgggg
     2821 gaggaggcag cggcggaggc ggcagcgggg gcggcggaag cgctaccggt aagcctatcc
     2881 ctaaccctct cctcggtctc gattctacgc gtaccggtat ggcttctaac tttactcagt
     2941 tcgttctcgt cgacaatggc ggaactggcg acgtgactgt cgccccaagc aacttcgcta
     3001 acggggtcgc tgaatggatc agctctaact cgcgatcaca ggcttacaaa gtaacctgta
     3061 gcgttcgtca gagctctgcg cagaatcgca aatacaccat caaagtcgag gtgcctaaag
     3121 tggcaaccca gactgttggt ggtgaagagc ttcctgtagc cggatggaga tcttacttaa
     3181 atatggaact aaccattcca attttcgcca cgaattccga ctgcgagctt attgttaagg
     3241 caatgcaagg tctcctaaaa gatggaaacc cgattccctc ggccatcgcg gccaactccg
     3301 gcatctacgg aggtggaggt ggatctcgta cgggttagta atgagtttaa acccgctgat
     3361 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     3421 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     3481 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     3541 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
     3601 aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat
     3661 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     3721 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     3781 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     3841 ccaaaaaact tgattagggt gatggttcac gtacctagaa gttcctattc cgaagttcct
     3901 attctctaga aagtatagga acttccttgg ccaaaaagcc tgaactcacc gcgacgtctg
     3961 tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag ctctcggagg
     4021 gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc ctgcgggtaa
     4081 atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt gcatcggccg
     4141 cgctcccgat tccggaagtg cttgacattg gggaattcag cgagagcctg acctattgca
     4201 tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg
     4261 ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc ggccgatctt agccagacga
     4321 gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg cgtgatttca
     4381 tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac gacaccgtca
     4441 gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac tgccccgaag
     4501 tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac aatggccgca
     4561 taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac gaggtcgcca
     4621 acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc tacttcgagc
     4681 ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc cgcattggtc
     4741 ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct tgggcgcagg
     4801 gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca caaatcgccc
     4861 gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat agtggaaacc
     4921 gacgccccag cactcgtccg agggcaaagg aatagcacgt actacgagat ttcgattcca
     4981 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     5041 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     5101 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     5161 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     5221 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     5281 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     5341 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     5401 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5461 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     5521 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     5581 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     5641 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     5701 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     5761 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     5821 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     5881 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5941 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     6001 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     6061 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     6121 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     6181 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     6241 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     6301 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     6361 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     6421 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     6481 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     6541 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     6601 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     6661 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     6721 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6781 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     6841 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6901 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6961 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     7021 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     7081 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     7141 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     7201 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     7261 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     7321 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     7381 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct
     7441 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc
     7501 tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca
     7561 aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg
     7621 cgatgtacgg gccagatata cgcgttgaca ttgattattg actaggcttt tgcaaaaagc
     7681 tttgcaaaga tggataaagt tttaaacaga gaggaatctt tgcagctaat ggaccttcta
     7741 ggtcttgaaa ggagtgggaa tt
//
