LOCUS       sequence-317343-        8101 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 8101)
  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 8101)
  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: CDC40
FEATURES             Location/Qualifiers
     source          1..8101
                     /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(3058..3082)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3207..3248
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3703..3720)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3709..3933
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4066..4085)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4217..4264
                     /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             4272..5294
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4272..5294
                     /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    5424..5545
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5461..5480)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5515..5534
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5594..5610)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5594..5610)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5607..5629)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5618..5634
                     /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(5642..5648,5649..5666,5667..5672))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5687..5708
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5825..5842)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5996..6584)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6076..6095)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6755..7546,7547..7615))
                     /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     7378..7397
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7616..7720)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7795..7814)
                     /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 gttggcacca
     1321 tgtcggctgc gattgcagct ctggccgctt cctatggttc gggttcaggg tccgaatcgg
     1381 actcggacag tgagagcagt cggtgtccgc tgccagccgc cgactccctc atgcacttga
     1441 ctaaatcgcc ttcatcaaag ccgtctctag cagtggcagt ggactcggct ccggaggtgg
     1501 cagttaagga agatttggag actggagttc accttgaccc tgccgtcaaa gaagttcagt
     1561 ataatcctac ctatgagacc atgtttgctc ctgagtttgg accagaaaat ccctttagga
     1621 cacagcaaat ggctgcccct agaaatatgc tttctggata tgccgaacca gctcatatca
     1681 atgatttcat gtttgagcag caaaggagaa cttttgcaac atatggttat gcattagacc
     1741 cttcattaga taatcatcaa gtgtctgcta aatatattgg ttctgtagaa gaagctgaaa
     1801 aaaatcaagg tttaactgta tttgaaactg gtcagaagaa aacagaaaag aggaaaaagt
     1861 ttaaagaaaa tgatgcatcc aatattgatg gttttttggg accatgggca aaatatgtgg
     1921 atgaaaaaga tgtagccaaa ccttcagaag aagagcaaaa agaattggat gaaatcacag
     1981 caaagaggca gaaaaaagga aaacaggaag aagagaaacc tggggaggag aagacaatct
     2041 tacatgttaa agaaatgtat gactatcaag gcaggtccta tcttcacata cctcaggatg
     2101 ttggtgttaa tctacggtca actatgccac ctgagaagtg ttatcttccc aaaaaacaaa
     2161 ttcatgtgtg gtctggacac acaaagggcg tcagtgcagt cagattgttt cctctctctg
     2221 gccatttatt gctgtcttgt tccatggact gtaaaattaa gctatgggag gtttatggag
     2281 aacggcgctg tctgagaaca tttattggtc acagtaaggc tgttagggat atctgcttca
     2341 atactgcagg aacacagttc ctcagtgcag cctatgacag gtatcttaag ctctgggaca
     2401 ctgagacagg acagtgtata tcaagattta caaaccgaaa agtaccttat tgtgtcaaat
     2461 tcaatcctga tgaagataag caaaatctct ttgtggctgg gatgtctgat aagaagattg
     2521 tgcaatggga cattcgaagt ggagaaattg tgcaggaata tgatcggcat ttgggagctg
     2581 tcaacaccat tgtttttgtg gatgagaata ggagatttgt gagcacatct gatgataaaa
     2641 gcctaagagt ttgggaatgg gatatccctg tggatttcaa gtacatagca gaacccagta
     2701 tgcactcaat gcctgcagtg actttgtctc caaatggaaa atggctagca tgccaatcaa
     2761 tggacaacca aatcttaatt tttggagcac agaacagatt tagattaaat aagaaaaaaa
     2821 tttttaaggg ccatatggta gcaggctatg cttgtcaggt ggacttttca ccagacatga
     2881 gttatgtgat ttcaggagat ggaaatggaa aattaaacat ttgggactgg aagaccacaa
     2941 aactctacag tcgatttaaa gctcatgata aagtgtgtat aggtgcagtg tggcatcctc
     3001 atgaaacttc taaggtcata acatgtggtt gggatggtct cattaaattg tgggatttgc
     3061 caactttctt gtacaaagtg gttgatatcc agcacagtgg cggccgctcg agtctagagg
     3121 gcccgcggtt cgaaaccagc ggaggcggcg gaagcggggg aggaggcagc ggcggaggcg
     3181 gcagcggggg cggcggaagc gctaccggta agcctatccc taaccctctc ctcggtctcg
     3241 attctacgcg taccggtatg gcttctaact ttactcagtt cgttctcgtc gacaatggcg
     3301 gaactggcga cgtgactgtc gccccaagca acttcgctaa cggggtcgct gaatggatca
     3361 gctctaactc gcgatcacag gcttacaaag taacctgtag cgttcgtcag agctctgcgc
     3421 agaatcgcaa atacaccatc aaagtcgagg tgcctaaagt ggcaacccag actgttggtg
     3481 gtgaagagct tcctgtagcc ggatggagat cttacttaaa tatggaacta accattccaa
     3541 ttttcgccac gaattccgac tgcgagctta ttgttaaggc aatgcaaggt ctcctaaaag
     3601 atggaaaccc gattccctcg gccatcgcgg ccaactccgg catctacgga ggtggaggtg
     3661 gatctcgtac gggttagtaa tgagtttaaa cccgctgatc agcctcgact gtgccttcta
     3721 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     3781 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     3841 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     3901 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg
     3961 gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     4021 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     4081 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     4141 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     4201 atggttcacg tacctagaag ttcctattcc gaagttccta ttctctagaa agtataggaa
     4261 cttccttggc caaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa
     4321 agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca
     4381 gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct
     4441 acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc
     4501 ttgacattgg ggaattcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg
     4561 tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg
     4621 ccatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac
     4681 cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc
     4741 atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc
     4801 tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg
     4861 atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga
     4921 gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt
     4981 ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag
     5041 gatcgccgcg gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct
     5101 tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc
     5161 gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga
     5221 ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga
     5281 gggcaaagga atagcacgta ctacgagatt tcgattccac cgccgccttc tatgaaaggt
     5341 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     5401 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     5461 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     5521 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     5581 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     5641 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     5701 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     5761 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     5821 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     5881 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     5941 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     6001 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     6061 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     6121 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     6181 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     6241 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     6301 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     6361 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     6421 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     6481 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     6541 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     6601 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     6661 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     6721 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     6781 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     6841 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     6901 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     6961 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     7021 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     7081 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     7141 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     7201 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     7261 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     7321 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     7381 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     7441 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     7501 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     7561 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     7621 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     7681 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     7741 cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat
     7801 ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc
     7861 tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat
     7921 gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac
     7981 gcgttgacat tgattattga ctaggctttt gcaaaaagct ttgcaaagat ggataaagtt
     8041 ttaaacagag aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat
     8101 t
//
