LOCUS       sequence-373710-        5598 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Destination vector for the expression of a fluorescent fusion of 
            N-ter mClover3 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 5598)
  TITLE     Lemaire Lab Plasmids 2022
REFERENCE   2  (bases 1 to 5598)
  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-mClover3-RfA
FEATURES             Location/Qualifiers
     source          1..5598
                     /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             join(2269..2271,2272..2274,2275..2985)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from mClover2, with improved photostability (Bajar et al., 
                     2003)"
                     /label=mClover3
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHYVYITADKQKNCIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(2314..2335)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2922..2943
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    2992..3116
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(3141..3146,3147..3164,3165..3171)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             3225..3884
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3291..3310)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             4226..4531
                     /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     4410..4429
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(4572..4696)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4954..4973)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4955..4973)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4980..4997)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4980..4996)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4989..5011)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(5137..5592)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5274..5295)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5486..5505
                     /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 ggtgagcaag
     2281 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     2341 ggccacaagt tcagcgtccg cggcgagggc gagggcgatg ccaccaacgg caagctgacc
     2401 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     2461 ttcggctacg gcgtggcctg cttcagccgc taccccgacc acatgaagca gcacgacttc
     2521 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatctcttt caaggacgac
     2581 ggtacctaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     2641 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     2701 aacttcaaca gccactacgt ctatatcacg gccgacaagc agaagaactg catcaaggct
     2761 aacttcaaga tccgccacaa cgttgaggac ggcagcgtgc agctcgccga ccactaccag
     2821 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagccat
     2881 cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     2941 gtgaccgccg ccgggattac acatggcatg gacgagctgt acaaggtaat cacaagtttg
     3001 tacaaaaaag ctgaacgaga aacgtaaaat gatataaata tcaatatatt aaattagatt
     3061 ttgcataaaa aacagactac ataatactgt aaaacacaac atatccagtc actatggcgg
     3121 ccgcattagg caccccaggc tttacacttt atgcttccgg ctcgtataat gtgtggattt
     3181 tgagttagga tccggcgaga ttttcaggag ctaaggaagc taaaatggag aaaaaaatca
     3241 ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt gaggcatttc
     3301 agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg gcctttttaa
     3361 agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt cttgcccgcc
     3421 tgatgaatgc tcatccggaa ttccgtatgg caatgaaaga cggtgagctg gtgatatggg
     3481 atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt tcatcgctct
     3541 ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa gatgtggcgt
     3601 gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg tttttcgtct
     3661 cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat atggacaact
     3721 tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag gtgctgatgc
     3781 cgctggcgat tcaggttcat catgccgtct gtgatggctt ccatgtcggc agaatgctta
     3841 atgaattaca acagtactgc gatgagtggc agggcggggc gtaaacgcgt ggatccggct
     3901 tactaaaagc cagataacag tatgcgtatt tgcgcgctga tttttgcggt ataagaatat
     3961 atactgatat gtatacccga agtatgtcaa aaagaggtgt gctatgaagc agcgtattac
     4021 agtgacagtt gacagcgaca gctatcagtt gctcaaggca tatatgatgt caatatctcc
     4081 ggtctggtaa gcacaaccat gcagaatgaa gcccgtcgtc tgcgtgccga acgctggaaa
     4141 gcggaaaatc aggaagggat ggctgaggtc gcccggttta ttgaaatgaa cggctctttt
     4201 gctgacgaga acagggactg gtgaaatgca gtttaaggtt tacacctata aaagagagag
     4261 ccgttatcgt ctgtttgtgg atgtacagag tgatattatt gacacgcccg ggcgacggat
     4321 ggtgatcccc ctggccagtg cacgtctgct gtcagataaa gtctcccgtg aactttaccc
     4381 ggtggtgcat atcggggatg aaagctggcg catgatgacc accgatatgg ccagtgtgcc
     4441 ggtctccgtt atcggggaag aagtggctga tctcagccac cgcgaaaatg acatcaaaaa
     4501 cgccattaac ctgatgttct ggggaatata aatgtcaggc tcccttatac acagccagtc
     4561 tgcaggtcga ccatagtgac tggatatgtt gtgttttaca gtattatgta gtctgttttt
     4621 tatgcaaaat ctaatttaat atattgatat ttatatcatt ttacgtttct cgttcagctt
     4681 tcttgtacaa agtggtgata aacttagata tcgcggccgc ggaagcgctg gttacctagg
     4741 aaccagcctc aagaacaccc gaatggagtc tctaagctac ataataccaa cttacacttt
     4801 acaaaatgtt gtcccccaaa atgtagccat tcgtatctgc yccwaawaaa aaraaarttt
     4861 yttcmcawtc waaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaamccccc cccccccccc
     4921 cctgcagggc caagtcggcc ggcgcgccat tcgccctata gtgagtcgta ttacaattca
     4981 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
     5041 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     5101 ccttcccaac agttgcgcag cctgaatggc gaatggaaat tgtaagcgtt aatattttgt
     5161 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     5221 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
     5281 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     5341 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
     5401 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     5461 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     5521 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     5581 tacagggcgc gtcaggtg
//
