LOCUS       sequence-373797-        5562 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Destination vector for the expression of a fluorescent fusion of 
            N-ter mEos4b and a protein of interest into embryos, by mRNA 
            microinjection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5562)
  TITLE     Lemaire Lab Plasmids 2022
REFERENCE   2  (bases 1 to 5562)
  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"
COMMENT     Sequence Label: pDEST-Spe4-Kozak-mEos4b-RfA
FEATURES             Location/Qualifiers
     source          1..5562
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        21..125
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(126..194,195..986)
                     /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(344..363)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1157..1745
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1646..1665
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1899..1916
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2033..2054
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2069..2074,2075..2092,2093..2099)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2107..2123
                     /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     2112..2134
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2131..2147
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2131..2147
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2168..2186
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     5'UTR           2211..2253
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     CDS             2269..2949
                     /codon_start=1
                     /product="fixation-resistant truly monomeric green-to-red 
                     photoswitchable fluorescent protein (Paez-Segala et al., 
                     2015)"
                     /label=mEos4b
                     /note="derived from mEos2"
                     /translation="MVSAIKPDMRIKLRMEGNVNGHHFVIDGDGTGKPYEGKQTMDLEV
                     KEGGPLPFAFDILTTAFHYGNRVFVKYPDNIQDYFKQSFPKGYSWERSLTFEDGGICNA
                     RNDITMEGDTFYNKVRFYGTNFPANGPVMQKKTLKWEPSTEKMYVRDGVLTGDIEMALL
                     LEGNAHYRCDFRTTYKAKEKGVKLPGAHFVDHAIEILSHDKDYNKVKLYEHAVAHSGLP
                     DNARR"
     protein_bind    2956..3080
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(3105..3110,3111..3128,3129..3135)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             3189..3848
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3255..3274)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             4190..4495
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     4374..4393
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(4536..4660)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4918..4937)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4919..4937)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4944..4961)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4944..4960)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4953..4975)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(5101..5556)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5238..5259)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5450..5469
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
       61 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
      121 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
      181 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
      241 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
      301 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
      361 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
      421 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
      481 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
      541 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
      601 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
      661 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
      721 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
      781 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
      841 gatctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
      901 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
      961 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     1021 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     1081 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     1141 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     1201 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     1261 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     1321 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     1381 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     1441 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     1501 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     1561 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     1621 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     1681 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     1741 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     1801 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     1861 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     1921 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     1981 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
     2041 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
     2101 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
     2161 agctcgaaat taaccctcac taaagggaac aaaagctgga gctaagcttg cttgttcttt
     2221 ttgcagaagc tcagaataaa cgctcaactt tggcagatct cagaaaaaat ggtgagtgcg
     2281 attaagccag acatgaggat caaactccgt atggaaggca acgtaaacgg gcaccacttt
     2341 gtgatcgacg gagatggtac aggcaagcct tatgagggaa aacagaccat ggatcttgaa
     2401 gtcaaagagg gcggacctct gccttttgcc tttgatatcc tgaccactgc attccattac
     2461 ggcaacaggg tattcgtgaa atatccagac aacatacaag actattttaa gcagtcgttt
     2521 cctaaggggt attcgtggga acgaagcttg actttcgaag acgggggcat ttgcaatgcc
     2581 agaaacgaca taacaatgga aggggacact ttctataata aagttcgatt ttatggtacc
     2641 aactttcccg ccaatggtcc agttatgcag aagaagacgc tgaaatggga gccctccact
     2701 gagaaaatgt atgtgcgtga tggagtgctg acgggtgata ttgagatggc tttgttgctt
     2761 gaaggaaatg cccattaccg atgtgacttc agaactactt acaaagctaa ggagaagggt
     2821 gtcaagttac caggcgccca ctttgtggac cacgccattg agattttaag ccatgacaaa
     2881 gattacaaca aggttaagct gtatgagcat gctgttgctc attctggatt gcctgacaat
     2941 gccagacgag taatcacaag tttgtacaaa aaagctgaac gagaaacgta aaatgatata
     3001 aatatcaata tattaaatta gattttgcat aaaaaacaga ctacataata ctgtaaaaca
     3061 caacatatcc agtcactatg gcggccgcat taggcacccc aggctttaca ctttatgctt
     3121 ccggctcgta taatgtgtgg attttgagtt aggatccggc gagattttca ggagctaagg
     3181 aagctaaaat ggagaaaaaa atcactggat ataccaccgt tgatatatcc caatggcatc
     3241 gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg tacctataac cagaccgttc
     3301 agctggatat tacggccttt ttaaagaccg taaagaaaaa taagcacaag ttttatccgg
     3361 cctttattca cattcttgcc cgcctgatga atgctcatcc ggaattccgt atggcaatga
     3421 aagacggtga gctggtgata tgggatagtg ttcacccttg ttacaccgtt ttccatgagc
     3481 aaactgaaac gttttcatcg ctctggagtg aataccacga cgatttccgg cagtttctac
     3541 acatatattc gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc cctaaagggt
     3601 ttattgagaa tatgtttttc gtctcagcca atccctgggt gagtttcacc agttttgatt
     3661 taaacgtggc caatatggac aacttcttcg cccccgtttt caccatgggc aaatattata
     3721 cgcaaggcga caaggtgctg atgccgctgg cgattcaggt tcatcatgcc gtctgtgatg
     3781 gcttccatgt cggcagaatg cttaatgaat tacaacagta ctgcgatgag tggcagggcg
     3841 gggcgtaaac gcgtggatcc ggcttactaa aagccagata acagtatgcg tatttgcgcg
     3901 ctgatttttg cggtataaga atatatactg atatgtatac ccgaagtatg tcaaaaagag
     3961 gtgtgctatg aagcagcgta ttacagtgac agttgacagc gacagctatc agttgctcaa
     4021 ggcatatatg atgtcaatat ctccggtctg gtaagcacaa ccatgcagaa tgaagcccgt
     4081 cgtctgcgtg ccgaacgctg gaaagcggaa aatcaggaag ggatggctga ggtcgcccgg
     4141 tttattgaaa tgaacggctc ttttgctgac gagaacaggg actggtgaaa tgcagtttaa
     4201 ggtttacacc tataaaagag agagccgtta tcgtctgttt gtggatgtac agagtgatat
     4261 tattgacacg cccgggcgac ggatggtgat ccccctggcc agtgcacgtc tgctgtcaga
     4321 taaagtctcc cgtgaacttt acccggtggt gcatatcggg gatgaaagct ggcgcatgat
     4381 gaccaccgat atggccagtg tgccggtctc cgttatcggg gaagaagtgg ctgatctcag
     4441 ccaccgcgaa aatgacatca aaaacgccat taacctgatg ttctggggaa tataaatgtc
     4501 aggctccctt atacacagcc agtctgcagg tcgaccatag tgactggata tgttgtgttt
     4561 tacagtatta tgtagtctgt tttttatgca aaatctaatt taatatattg atatttatat
     4621 cattttacgt ttctcgttca gctttcttgt acaaagtggt gataaactta gatatcgcgg
     4681 ccgcggaagc gctggttacc taggaaccag cctcaagaac acccgaatgg agtctctaag
     4741 ctacataata ccaacttaca ctttacaaaa tgttgtcccc caaaatgtag ccattcgtat
     4801 ctgctccwaa waaaaaraaa rtttyttcmc awtcwaaaaa aaaaaaaaaa aaaaaaaaaa
     4861 aaaaaaaamm cccccccccc cccccctgca gggccaagtc ggccggcgcg ccattcgccc
     4921 tatagtgagt cgtattacaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     4981 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     5041 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     5101 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     5161 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     5221 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     5281 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
     5341 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     5401 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     5461 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     5521 cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg tg
//
