LOCUS       sequence-373834-        5583 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Destination vector for the expression of a fluorescent fusion of a 
            protein of interest with C-ter mClover3 into embryos, by mRNA 
            microinjection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5583)
  TITLE     Lemaire Lab Plasmids 2022
REFERENCE   2  (bases 1 to 5583)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pDEST-Spe4-RfA-mClover3-stop
FEATURES             Location/Qualifiers
     source          1..5583
                     /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"
     protein_bind    2282..2406
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(2431..2436,2437..2454,2455..2461)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             2515..3174
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2581..2600)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             3516..3821
                     /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     3700..3719
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(3862..3986)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             join(4000..4002,4003..4005,4006..4719)
                     /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(4045..4066)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     4653..4674
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     3'UTR           4726..4851
                     /label=Xenopus globin 3'-UTR
                     /note="3'-UTR of the major beta-globin gene of Xenopus 
                     laevis"
     primer_bind     complement(4939..4958)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4940..4958)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4965..4982)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4965..4981)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4974..4996)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(5122..5577)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5259..5280)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5471..5490
                     /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 gacgtcaggc cttacgtaat
     2281 cacaagtttg tacaaaaaag ctgaacgaga aacgtaaaat gatataaata tcaatatatt
     2341 aaattagatt ttgcataaaa aacagactac ataatactgt aaaacacaac atatccagtc
     2401 actatggcgg ccgcattagg caccccaggc tttacacttt atgcttccgg ctcgtataat
     2461 gtgtggattt tgagttagga tccggcgaga ttttcaggag ctaaggaagc taaaatggag
     2521 aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt
     2581 gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg
     2641 gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt
     2701 cttgcccgcc tgatgaatgc tcatccggaa ttccgtatgg caatgaaaga cggtgagctg
     2761 gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt
     2821 tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa
     2881 gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg
     2941 tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat
     3001 atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag
     3061 gtgctgatgc cgctggcgat tcaggttcat catgccgtct gtgatggctt ccatgtcggc
     3121 agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc gtaaacgcgt
     3181 ggatccggct tactaaaagc cagataacag tatgcgtatt tgcgcgctga tttttgcggt
     3241 ataagaatat atactgatat gtatacccga agtatgtcaa aaagaggtgt gctatgaagc
     3301 agcgtattac agtgacagtt gacagcgaca gctatcagtt gctcaaggca tatatgatgt
     3361 caatatctcc ggtctggtaa gcacaaccat gcagaatgaa gcccgtcgtc tgcgtgccga
     3421 acgctggaaa gcggaaaatc aggaagggat ggctgaggtc gcccggttta ttgaaatgaa
     3481 cggctctttt gctgacgaga acagggactg gtgaaatgca gtttaaggtt tacacctata
     3541 aaagagagag ccgttatcgt ctgtttgtgg atgtacagag tgatattatt gacacgcccg
     3601 ggcgacggat ggtgatcccc ctggccagtg cacgtctgct gtcagataaa gtctcccgtg
     3661 aactttaccc ggtggtgcat atcggggatg aaagctggcg catgatgacc accgatatgg
     3721 ccagtgtgcc ggtctccgtt atcggggaag aagtggctga tctcagccac cgcgaaaatg
     3781 acatcaaaaa cgccattaac ctgatgttct ggggaatata aatgtcaggc tcccttatac
     3841 acagccagtc tgcaggtcga ccatagtgac tggatatgtt gtgttttaca gtattatgta
     3901 gtctgttttt tatgcaaaat ctaatttaat atattgatat ttatatcatt ttacgtttct
     3961 cgttcagctt tcttgtacaa agtggtgata aacttagata tggtgagcaa gggcgaggag
     4021 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     4081 ttcagcgtcc gcggcgaggg cgagggcgat gccaccaacg gcaagctgac cctgaagttc
     4141 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cttcggctac
     4201 ggcgtggcct gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     4261 gccatgcccg aaggctacgt ccaggagcgc accatctctt tcaaggacga cggtacctac
     4321 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     4381 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caacttcaac
     4441 agccactacg tctatatcac ggccgacaag cagaagaact gcatcaaggc taacttcaag
     4501 atccgccaca acgttgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     4561 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcca tcagtccaag
     4621 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     4681 gccgggatta cacatggcat ggacgagctg tacaagtgac ctaggaacca gcctcaagaa
     4741 cacccgaatg gagtctctaa gctacataat accaacttac actttacaaa atgttgtccc
     4801 ccaaaatgta gccattcgta tctgctccta awaaaaaraa artttcttca cattctaaaa
     4861 aaaaaaaaaa aaaaaaaaaa aaaaaaaaam mccccccccc ccccccctgc agggccaagt
     4921 cggccggcgc gccattcgcc ctatagtgag tcgtattaca attcactggc cgtcgtttta
     4981 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
     5041 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     5101 cgcagcctga atggcgaatg gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
     5161 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     5221 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     5281 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     5341 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     5401 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     5461 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     5521 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcag
     5581 gtg
//
