LOCUS       sequence-373805-        5589 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Destination vector for the expression of a fluorescent fusion of 
            N-ter mNeonGreen 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 5589)
  TITLE     Lemaire Lab Plasmids 2022
REFERENCE   2  (bases 1 to 5589)
  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-mNeonGreen-RfA
FEATURES             Location/Qualifiers
     source          1..5589
                     /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..2976
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      2524..2533
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2983..3107
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(3132..3137,3138..3155,3156..3162)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             3216..3875
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3282..3301)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             4217..4522
                     /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     4401..4420
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(4563..4687)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4945..4964)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4946..4964)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4971..4988)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4971..4987)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4980..5002)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(5128..5583)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5265..5286)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5477..5496
                     /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 ggcgaggagg ataacatggc ctctctccca gcgacacatg agttacacat ctttggctcg
     2341 atcaacggtg tggactttga catggtgggt cagggcaccg gcaatccaaa tgatggttat
     2401 gaggagttaa acctgaagtc caccaagggt gacctccagt tctccccctg gattctggtc
     2461 cctcatatcg ggtatggctt ccatcagtac ctgccctacc ctgacgggat gtcgcctttc
     2521 caggccgcca tggtagatgg ctccggatac caagtccatc gcacaatgca gtttgaagat
     2581 ggtgcctccc ttactgttaa ctaccgctac acctacgagg gaagccacat caaaggagag
     2641 gcccaggtga aggggactgg tttccctgct gacggtcctg tgatgaccaa ctcgctgacc
     2701 gctgcggact ggtgcaggtc gaagaagact taccccaacg acaaaaccat catcagtacc
     2761 tttaagtgga gttacaccac tggaaatggc aagcgctacc ggagcactgc gcggaccacc
     2821 tacacctttg ccaagccaat ggcggctaac tatctgaaga accagccgat gtacgtgttc
     2881 cgtaagacgg agctcaagca ctccaagacc gagctcaact tcaaggagtg gcaaaaggcc
     2941 tttaccgatg tgatgggcat ggacgagctg tacaaggtaa tcacaagttt gtacaaaaaa
     3001 gctgaacgag aaacgtaaaa tgatataaat atcaatatat taaattagat tttgcataaa
     3061 aaacagacta cataatactg taaaacacaa catatccagt cactatggcg gccgcattag
     3121 gcaccccagg ctttacactt tatgcttccg gctcgtataa tgtgtggatt ttgagttagg
     3181 atccggcgag attttcagga gctaaggaag ctaaaatgga gaaaaaaatc actggatata
     3241 ccaccgttga tatatcccaa tggcatcgta aagaacattt tgaggcattt cagtcagttg
     3301 ctcaatgtac ctataaccag accgttcagc tggatattac ggccttttta aagaccgtaa
     3361 agaaaaataa gcacaagttt tatccggcct ttattcacat tcttgcccgc ctgatgaatg
     3421 ctcatccgga attccgtatg gcaatgaaag acggtgagct ggtgatatgg gatagtgttc
     3481 acccttgtta caccgttttc catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat
     3541 accacgacga tttccggcag tttctacaca tatattcgca agatgtggcg tgttacggtg
     3601 aaaacctggc ctatttccct aaagggttta ttgagaatat gtttttcgtc tcagccaatc
     3661 cctgggtgag tttcaccagt tttgatttaa acgtggccaa tatggacaac ttcttcgccc
     3721 ccgttttcac catgggcaaa tattatacgc aaggcgacaa ggtgctgatg ccgctggcga
     3781 ttcaggttca tcatgccgtc tgtgatggct tccatgtcgg cagaatgctt aatgaattac
     3841 aacagtactg cgatgagtgg cagggcgggg cgtaaacgcg tggatccggc ttactaaaag
     3901 ccagataaca gtatgcgtat ttgcgcgctg atttttgcgg tataagaata tatactgata
     3961 tgtatacccg aagtatgtca aaaagaggtg tgctatgaag cagcgtatta cagtgacagt
     4021 tgacagcgac agctatcagt tgctcaaggc atatatgatg tcaatatctc cggtctggta
     4081 agcacaacca tgcagaatga agcccgtcgt ctgcgtgccg aacgctggaa agcggaaaat
     4141 caggaaggga tggctgaggt cgcccggttt attgaaatga acggctcttt tgctgacgag
     4201 aacagggact ggtgaaatgc agtttaaggt ttacacctat aaaagagaga gccgttatcg
     4261 tctgtttgtg gatgtacaga gtgatattat tgacacgccc gggcgacgga tggtgatccc
     4321 cctggccagt gcacgtctgc tgtcagataa agtctcccgt gaactttacc cggtggtgca
     4381 tatcggggat gaaagctggc gcatgatgac caccgatatg gccagtgtgc cggtctccgt
     4441 tatcggggaa gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa acgccattaa
     4501 cctgatgttc tggggaatat aaatgtcagg ctcccttata cacagccagt ctgcaggtcg
     4561 accatagtga ctggatatgt tgtgttttac agtattatgt agtctgtttt ttatgcaaaa
     4621 tctaatttaa tatattgata tttatatcat tttacgtttc tcgttcagct ttcttgtaca
     4681 aagtggtgat aaacttagat atcgcggccg cggaagcgct ggttacctag gaaccagcct
     4741 caagaacacc cgaatggagt ctctaagcta cataatacca acttacactt tacaaaatgt
     4801 tgtcccccaa aatgtagcca ttcgtatctg ctcctaawaa aaaraaartt tyttcacatt
     4861 cwaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaammccc cccccccccc ccctgcaggg
     4921 ccaagtcggc cggcgcgcca ttcgccctat agtgagtcgt attacaattc actggccgtc
     4981 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     5041 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     5101 cagttgcgca gcctgaatgg cgaatggaaa ttgtaagcgt taatattttg ttaaaattcg
     5161 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     5221 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
     5281 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     5341 atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     5401 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     5461 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     5521 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     5581 cgtcaggtg
//
