LOCUS       sequence-378128-        8099 bp    DNA     circular SYN 14-APR-2025
DEFINITION  EB3 tagged with mScarlet-I, expressed under the UAS promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8099)
  AUTHORS   Moller K, Brambach M, Villani A, Gallo E, Gilmour D, Peri F
  TITLE     A role for the centrosome in regulating the rate of neuronal 
            efferocytosis by microglia in vivo.
  JOURNAL   Elife. 2022 Nov 18;11:e82094. doi: 10.7554/eLife.82094.
  PUBMED    36398880
REFERENCE   2  (bases 1 to 8099)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Elife."; 
            date: "2022-11-18"; volume: "11:e82094. doi"; pages: " 
            10.7554/eLife.82094"
COMMENT     Sequence Label: pDEST_5xUAS:EB3-mScarlet-I
FEATURES             Location/Qualifiers
     source          1..8099
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    62..196
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    198..222
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(227..922)
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(502..521)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     771..789
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      919..928
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(1789..1813)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    1893..1987
                     /label=5X UAS
                     /bound_moiety="GAL4"
                     /note="five tandem copies of the ""ScaI site"" 17-mer 
                     CGGAGTACTGTCCTCCG, an upstream activating sequence (UAS) 
                     that efficiently binds yeast Gal4 (Webster et al., 1988; 
                     Pfeiffer et al., 2010)"
     primer_bind     complement(2003..2019)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(2030..2050)
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     complement(2070..2086)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2982..3003
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3166..3270
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3271..3339,3340..4131)
                     /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     complement(3489..3508)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4302..4890
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4791..4810
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5044..5061
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5178..5199
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5214..5219,5220..5237,5238..5244)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5252..5268
                     /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)."
     primer_bind     5257..5279
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5276..5292
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5311..5331
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5313..5331
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     5368..5384
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(6597..6630)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     regulatory      6783..6792
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(6789..6791,6792..6794,6795..7508)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6834..6855)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(7095..7114)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7442..7463
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(7531..7548)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7537..7761
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    7904..8038
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     8054..8070
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    8078..8098
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
ORIGIN
        1 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt
       61 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
      121 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
      181 ttatcatgtc tggatccccc actttgtaca agaaagctgg gtcttacttg tacagctcgt
      241 ccatgccgcc ggtggagtgg cggccctcgg agcgttcgta ctgttccacc acggtgtagt
      301 cctcgttgtg ggaggtgatg tccaacttgc ggtcgacgtt gtaggcgccg ggcatctgca
      361 cgggcttctt ggccttgtag gtggtcttga agtccgccag gtagcggccg ccgtccttca
      421 ggcgcagggc catcttaatg tcgcccttca gcacgccgtc ctcggggtac aaccgctcgg
      481 tggacgcttc ccagcccatt gtcttcttct gcattacggg gccgtcagga gggaagttgg
      541 tgccgcggag cttcaccttg tagatcaggg tgccgtcctc cagggaggtg tcctgggtca
      601 cggtcacggc gccgccgtcc tcgaagttca tcacgcgctc ccacttgaag ccctcgggga
      661 aggactgctt atagtagtcg gggatgtcgg cggggtgctt gatgaaggcc ctggagccgt
      721 acatgaactg aggggacagg atgtcccagg agaagggcag ggggccaccc ttggtcacct
      781 tcagcttggc ggtctgggtg ccctcgtagg ggcggccctc gccctcgccc tcgatctcga
      841 actcgtggcc gttcatggag ccctccatgt gcaccttgaa ccgcatgaac tccttgatca
      901 ctgcctcgcc cttgctcacc atggtggcga ccggtggatc caggtactcg tcctggtctt
      961 cttgttgatg ctcttcaatc tcatcgtcct cagggggtgc gaatccttcc tctgtggcat
     1021 agaggatgcc aatgatgcct gagataacag ggctgttttc actttcatgc tcctggcaga
     1081 tgagctcgat gtcacgaagt ttgctgaagt agaagtcacg ttccttctcc agcccatcca
     1141 ctgtcagctt caagtccacc agctgttggt tgagttcaag aatttgggca tcagtctcat
     1201 ggccgccatt tcgggctgat ggaggattct tccggagaat gcaggggggg gccacattgc
     1261 tcagccggcc agaggtctgc atgttttttg ggcctgtggg ggacgtcctc tgtggaactg
     1321 ctgtgccaat gagtttcttg gatttgttga agatctgatc acctgggtta ggaggtggcg
     1381 ctacgtcctg gccctgccgc gccagcagag ggttgtaatc ctttccatca tagtttgcgt
     1441 caaagaattt cttaaaccac tgaataaact caaaattatc ttggaatttt cctttcacta
     1501 atttctctac aggaatgatt ttgtcaacac ccatcttctt gaaagctgct tgcagcacct
     1561 tgaagttgtg gatgtattca tgctctagtt tggcctggaa cttcactttc ctcaagtgca
     1621 cacagccggg gaagagcatg tccatgaact ggcagtaggc tgcccctgaa caaagctgtt
     1681 ctatcttggt atagttgagg tgcagggagt cgttgaccca tgcaagcata tcatggcgac
     1741 tcagattttc actggtcaca gatgtggagt acacattgac ggccatccag cctgcttttt
     1801 tgtacaaact tgcggtggcg gcgaattcgt ggaggagctc aaagtgaggc tgagacgcga
     1861 tgggatccat tatataccct ctagagtctc cgctcggagg acagtactcc gctcggagga
     1921 cagtactccg ctcggaggac agtactccgc tcggaggaca gtactccgct cggaggacag
     1981 tactccgacc tgcagcccgg gggatccact agttctagag cggccgccgc aacttttcta
     2041 tacaaagttg atagcttggc gtaatcatgg tcatagctgt ttcctgctcg agggcccatc
     2101 tggcctgtgt ttcagacacc agggagtctc tgctcacgtt tcctgctatt tgcagcctct
     2161 ctatcaagac taatacacct cttcccgcat cggctgcctg tgagaggctt ttcagcactg
     2221 caggattgct tttcagcccc aaaagagcta ggcttgacac taacaatttt gagaatcagc
     2281 ttctactgaa gttaaatctg aggttttaca actttgagta gcgtgtactg gcattagatt
     2341 gtctgtctta tagtttgata attaaataca aacagttcta aagcaggata aaaccttgta
     2401 tgcatttcat ttaatgtttt ttgagattaa aagcttaaac aagaatctct agttttcttt
     2461 cttgctttta cttttacttc cttaatactc aagtacaatt ttaatggagt acttttttac
     2521 ttttactcaa gtaagattct agccagatac ttttactttt aattgagtaa aattttccct
     2581 aagtacttgt actttcactt gagtaaaatt tttgagtact ttttacacct ctgtcaagaa
     2641 ctcctggaca aacctctgac ctgtgtggaa cagagtggat atgggtgtct gaacagatat
     2701 tcacgtcttt tgcagatcag agggcatttc tggtgttgtg caatgacctg ggtccaacag
     2761 gtaggcgtac tcgtatccaa gagcattgtt ggacaggagg tagtctccat tcctataaag
     2821 aggaaggaat ggcaccatgt agtagccgtc attgtggcca attggtgcat tggtacgtgg
     2881 gtagatggac cggggaggct gatgagttct tagccagcgt gggccatcgc cctgatagac
     2941 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     3001 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     3061 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     3121 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     3181 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     3241 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     3301 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     3361 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     3421 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     3481 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     3541 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     3601 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     3661 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     3721 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     3781 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     3841 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     3901 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     3961 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     4021 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     4081 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     4141 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     4201 taggtgaaga tcctttttga taatctcatg accaaaatgc cttaacgtga gttttcgttc
     4261 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     4321 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     4381 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     4441 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     4501 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     4561 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     4621 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     4681 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     4741 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     4801 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     4861 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     4921 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     4981 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     5041 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
     5101 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     5161 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac
     5221 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga
     5281 aacagctatg accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag
     5341 ctggagctcc accgcggtgg cggccgctct agaactagtg gatctgctgg gcttgctgaa
     5401 ggtagggggt caagaaccag aggtgtaaag tacttgagta attttacttg attactgtac
     5461 ttaagtatta tttttgggga tttttacttt acttgagtac aattaaaaat caatactttt
     5521 acttttactt aattacattt ttttagaaaa aaaagtactt tttactcctt acaattttat
     5581 ttacagtcaa aaagtactta ttttttggag atcacttcat tctattttcc cttgctatta
     5641 ccaaaccaat tgaattgcgc tgatgcccag tttaatttaa atgttattta ttctgcctat
     5701 gaaaatcgtt ttcacattat atgaaattgg tcagacatgt tcattggtcc tttggaagtg
     5761 acgtcatgtc acatctatta ccacaatgca cagcaccttg acctggaaat tagggaaatt
     5821 ataacagtca atcagtggaa gaaaatggag gaagtatgtg attcatcagc agctgcgagc
     5881 agcacagtcc aaaatcagcc acaggatcaa gagcacccgt ggccgtatct tcgcagatcg
     5941 ggccccccct cgagcaattg gtgctatcat catgacaaag agagaagaaa tcagttatta
     6001 gaaatgagtt attaaaacta ttatgattag aaatatgttt ctttctctta aacaggattt
     6061 aaggggaaaa tatacaggga gcgaaaaatt ttgacttcat ctgtatatac atataataaa
     6121 taatacatca ataaattcca tctaaatacg ctaccatata tatatatata tgtatataca
     6181 cacacacaca cacaccatct aatatatgtc atgctacatt attaacttca gtatgaaatc
     6241 tactctgctc tggagtgtat cagcgagcga ctcctctgct cggattatca tcacgtgacc
     6301 tctgacctga acacagacct ctcttagcca taggtataca gcctatagca tagagctcgg
     6361 gctgagaggg aaagtgccgc cgtcagggct ttccattgac agtaaaatgc ttacggcctt
     6421 ctgggcgatg cgatgaccct gcgcttcggc acggctgaac tctgatgacg gcgtgtttct
     6481 attgtccgcc gcgcgccgct gtgctgccct tgccagcgta taaaagctgc ggcagtggtg
     6541 aggaaggcca gagcgagcca gccttcccag cacagctgta gtttaaacgc gttgccataa
     6601 cttcgtatag gatactttat acgaagttat ctgcatgcta ggttgccggg aacggtgcat
     6661 tggaacgcgc attccccgtg ttaattaaca ggtaagtgtc ttcctcctgt ttccttcccc
     6721 tgctattctg ctcaaccttc ctatcagaaa ctgcagtatc tgtatttttg ctaggggatc
     6781 ccgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     6841 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     6901 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     6961 ggcccaccct cgtgaccacc ctgacctggg gcgtgcagtg cttcagccgc taccccgacc
     7021 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     7081 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     7141 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     7201 tggggcacaa gctggagtac aactacatca gccacaacgt ctatatcacc gccgacaagc
     7261 agaagaacgg catcaaggcc aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
     7321 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     7381 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
     7441 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     7501 acaagtaaag cggccaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat
     7561 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     7621 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     7681 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     7741 gggatgcggt gggctctatg gcttctgagg cggaaagaac caggatctga tctagaggat
     7801 cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct
     7861 ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc
     7921 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     7981 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatcat
     8041 catcgatggt accgtaaaac gacggccagt gaattatcaa ctatgtataa taaagttgg
//
