LOCUS       sequence-376716-        7627 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 7627)
  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 7627)
  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: RBM22
FEATURES             Location/Qualifiers
     source          1..7627
                     /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(2584..2608)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2733..2774
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3229..3246)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3235..3459
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3592..3611)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3743..3790
                     /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             3798..4820
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3798..4820
                     /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    4950..5071
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4987..5006)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5041..5060
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5120..5136)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5120..5136)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5133..5155)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5144..5160
                     /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(5168..5174,5175..5192,5193..5198))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5213..5234
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5351..5368)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5522..6110)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5602..5621)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6281..7072,7073..7141))
                     /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     6904..6923
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7142..7246)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7321..7340)
                     /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 gctggcacca
     1321 tggcgacctc tctgggttcc aacacctaca acaggcagaa ctgggaggat gcggacttcc
     1381 ccattctgtg ccagacatgt cttggagaaa acccatatat ccgaatgacc aaagaaaagt
     1441 atgggaagga atgcaaaatc tgtgccaggc cattcacagt gtttcgctgg tgccctggag
     1501 tccgcatgcg tttcaagaag actgaagtgt gccaaacctg cagtaaattg aagaatgtct
     1561 gtcagacctg cctcttagac ctagagtatg gcctgcccat ccaggttcgt gacgcaggat
     1621 tgtcttttaa agatgacatg ccaaagtcag atgtcaacaa agagtactat acacagaata
     1681 tggagagaga gatttctaac tctgatggaa cacggccagt tggcatgctg gggaaagcca
     1741 catctaccag tgacatgctg ctcaaactgg cccggaccac accctactac aaaaggaatc
     1801 gaccccacat ttgctccttc tgggtgaaag gagagtgtaa gagaggagag gaatgtccat
     1861 acagacatga gaagcctaca gatccagatg acccccttgc tgatcagaat attaaagacc
     1921 gttattacgg aatcaatgat cctgtagctg acaagcttct aaagcgggct tcaacaatgc
     1981 ctcggctgga cccaccagag gataaaacta tcaccacact atatgttggt ggtctaggtg
     2041 ataccattac tgagacagat ttaagaaatc atttctacca gttcggagag atccggacga
     2101 tcactgttgt gcagagacag cagtgtgctt tcatccagtt tgccacacgg caggctgcag
     2161 aagtggctgc tgagaagtcc tttaataagt tgattgtaaa tggccgcaga ctgaatgtga
     2221 aatggggaag atcccaggca gccagaggaa aagaaaaaga gaaagatgga actacagact
     2281 ctgggatcaa actagaacct gttccaggat tgccaggagc tcttcctcct cctcctgcag
     2341 cagaagaaga agcctctgcc aactacttca acttgccccc aagtggtcct ccagctgtgg
     2401 tgaacattgc tctgccaccg ccccctggca ttgctccacc cccaccccca ggttttgggc
     2461 cacacatgtt ccacccaatg ggaccacccc ctcctttcat gcgggctcca ggaccaatcc
     2521 actatccttc tcaggaccct cagaggatgg gagctcatgc tggaaaacac agcagccccg
     2581 gcgacccagc tttcttgtac aaagtggttg atatccagca cagtggcggc cgctcgagtc
     2641 tagagggccc gcggttcgaa accagcggag gcggcggaag cgggggagga ggcagcggcg
     2701 gaggcggcag cgggggcggc ggaagcgcta ccggtaagcc tatccctaac cctctcctcg
     2761 gtctcgattc tacgcgtacc ggtatggctt ctaactttac tcagttcgtt ctcgtcgaca
     2821 atggcggaac tggcgacgtg actgtcgccc caagcaactt cgctaacggg gtcgctgaat
     2881 ggatcagctc taactcgcga tcacaggctt acaaagtaac ctgtagcgtt cgtcagagct
     2941 ctgcgcagaa tcgcaaatac accatcaaag tcgaggtgcc taaagtggca acccagactg
     3001 ttggtggtga agagcttcct gtagccggat ggagatctta cttaaatatg gaactaacca
     3061 ttccaatttt cgccacgaat tccgactgcg agcttattgt taaggcaatg caaggtctcc
     3121 taaaagatgg aaacccgatt ccctcggcca tcgcggccaa ctccggcatc tacggaggtg
     3181 gaggtggatc tcgtacgggt tagtaatgag tttaaacccg ctgatcagcc tcgactgtgc
     3241 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     3301 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     3361 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     3421 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca
     3481 gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     3541 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     3601 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     3661 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     3721 agggtgatgg ttcacgtacc tagaagttcc tattccgaag ttcctattct ctagaaagta
     3781 taggaacttc cttggccaaa aagcctgaac tcaccgcgac gtctgtcgag aagtttctga
     3841 tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa gaatctcgtg
     3901 ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg
     3961 gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg
     4021 aagtgcttga cattggggaa ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac
     4081 agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg
     4141 cggaggccat ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat
     4201 tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg
     4261 atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc
     4321 aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc
     4381 acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg
     4441 actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga
     4501 ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc
     4561 ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc
     4621 agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa
     4681 tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg
     4741 tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc
     4801 gtccgagggc aaaggaatag cacgtactac gagatttcga ttccaccgcc gccttctatg
     4861 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     4921 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     4981 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     5041 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     5101 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     5161 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     5221 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     5281 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     5341 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     5401 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     5461 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     5521 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     5581 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     5641 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     5701 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     5761 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     5821 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     5881 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     5941 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     6001 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     6061 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     6121 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     6181 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     6241 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     6301 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     6361 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     6421 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     6481 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     6541 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     6601 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     6661 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     6721 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     6781 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     6841 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     6901 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     6961 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     7021 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     7081 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     7141 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     7201 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     7261 aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag
     7321 tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga
     7381 ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa
     7441 ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag
     7501 atatacgcgt tgacattgat tattgactag gcttttgcaa aaagctttgc aaagatggat
     7561 aaagttttaa acagagagga atctttgcag ctaatggacc ttctaggtct tgaaaggagt
     7621 gggaatt
//
