LOCUS       sequence-487154-        8323 bp    DNA     circular SYN 25-SEP-2025
DEFINITION  Zebrafish transgenesis plasmid encoding cytoplasmic mNeonGreen-NES 
            and mKate2-NLS separated by the P2A peptide, driven by the QUAS 
            enhancer, and including a cmlc2:mKate2 cardiac transgenesis marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8323)
  TITLE     Fibroblasts promote osmotic surveillance by wound-induced unique 
            calcium patterns
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8323)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 25, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pDestTol2CR-QUAS:mNeonGreen-NES-P2A-mKate2-NLS
FEATURES             Location/Qualifiers
     source          1..8323
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(21..37)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    53..187
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    309..533
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     522..539
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     regulatory      1350..1359
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2815..2831)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2815..2831)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2868..2888)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2868..2886)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2907..2923)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2920..2942)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2931..2947
                     /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)."
     promoter        complement(join(2955..2961,2962..2979,2980..2985))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3000..3021
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3138..3155)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3309..3897)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3389..3408)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4068..4859,4860..4928))
                     /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     4691..4710
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4929..5033)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5196..5217)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6113..6129
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6149..6169
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    6360..6384
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      6402..6411
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6408..7115
                     /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      6663..6672
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(7159..7183)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             7194..7250
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             7251..7946
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHR"
     CDS             7992..8012
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8016..8036
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8040..8060
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    8119..8253
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(join(8316..8323,1..13))
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
ORIGIN
        1 ttatacatag ttgataattc actggccgtc gttttacggt accatcgatg atgatccaga
       61 catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg
      121 ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa
      181 acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga
      241 ggttttttaa agcaagtaaa acctctacaa atgtggtatg gctgattatg atcctctaga
      301 tcagatctcc atagagccca ccgcatcccc agcatgcctg ctattgtctt cccaatcctc
      361 ccccttgctg tcctgcccca ccccaccccc cagaatagaa tgacacctac tcagacaatg
      421 cgatgcaatt tcctcatttt attaggaaag gacagtggga gtggcacctt ccagggtcaa
      481 ggaaggcacg ggggaggggc aaacaacaga tggctggcaa ctagaaggca cagtcgaggc
      541 tgatcagcgg gtttaaacgg gccctctaga ctcgagcggc cgccactgtg ctggatatct
      601 gcagaattca cctgctcctc ctgctcccgc tgctgcgcct ccggacagtt tgtgccccag
      661 tttgctaggg aggtcgcagt atctggccac agccacctcg tgctgctcga cgtaggtctc
      721 tttgtcggcc tccttgattc tttccagtct tctgtccaca tagtagacgc cgggcatctt
      781 gaggttctta gcgggtttct tggatctgta tgtggtcttc aagttgcaga tcaggtggcc
      841 cccgcccacg agcttcaggg ccatgtcggc tctgccttcc aggccgccgt cagcggggta
      901 cagggtctcg gtggaggcct cccagccgag tgttttcttc tgcatcacag ggccgttgga
      961 tgggaagttc acccctctga tcttgacgtt gtagatgagg cagccgtcct ggaggctggt
     1021 gtcctgggta gcggtcagca cgcccccgtc ttcgtatgtg gtgactctct cccatgtgaa
     1081 gccctcaggg aaggactgct taaagaagtc ggggatgccc tgggtgtggt tgatgaaggt
     1141 tttgctgccg tacatgaagc tggtagccag gatgtcgaag gcgaagggga gagggccgcc
     1201 ctcgacggcc ttgattctca tggtctgggt gccctcgtag ggcttgcctt cgccctcgga
     1261 tgtgcacttg aagtggtggt tgttcacggt gccctccatg tacagcttca tgtgcatgtt
     1321 ctccttaatc agctcttcgc ccttagacac catggtggcg gtcactgtct gctttgctgt
     1381 tggtctgggc tcctgggtca ctggcttact aatggagtct ttatgtatga ggactcttat
     1441 caattgttct tctataaagg tctgcagtgt ttctgttcgt cccctacatg gacacccaga
     1501 gcctcctaaa tacaggagcc ctgataactg cacaagtgct cagattccag cagggtggaa
     1561 aatgagataa agtgtgcaga tggggagggg gacgtgaatg agagatttga gggatgaaaa
     1621 ggatggatga acgcattgaa aatagcccct ttcacaaaat aataccagta aattgtcata
     1681 taattaatga ccagaccttt acctgtaaat gatgtgatgt tcatatacag aagaatgcca
     1741 acaatttaca gatgatttta caaattctta attttccttt tctaaatgga tttgttcaca
     1801 cattatctct tcaaagcaat tgaattttct ggaaaagatt ggctgtgtgt gtaaaaggtg
     1861 ctactgagga atttaatgtg acatggaatg aagcaaacag cagcctaaga caggatggca
     1921 ggaaaaatgt cacatgttta aaataagagt gaaaccaacg acctggactt atagagactg
     1981 tgtgtcctta ggcaaagctc ttgtacaaac agtgcttgtg actgtgaaat tcggggtttg
     2041 cctggattgt gttaaatgtg tgcttggatg tatcataaaa aagaatcggt tctctgtatt
     2101 tgacattttc tgtaatttat ctaaagttac aactggctat gcctgatttt tatcactatt
     2161 gaatcaatca ttttaaagaa tgttaagtct gctgatttgg agagctcatt tacatttatt
     2221 ttgaatgtct cttatttaac acaactgatt taagctttag atctgcgaag atacggccac
     2281 gggtgctctt gatcctgtgg ctgattttgg actgtgctgc tcgcagctgc tgatgaatca
     2341 catacttcct ccattttctt ccactgattg actgttataa tttccctaat ttccaggtca
     2401 aggtgctgtg cattgtggta atagatgtga catgacgtca cttccaaagg accaatgaac
     2461 atgtctgacc aatttcatat aatgtgaaaa cgattttcat aggcagaata aataacattt
     2521 aaattaaact gggcatcagc gcaattcaat tggtttggta atagcaaggg aaaatagaat
     2581 gaagtgatct ccaaaaaata agtacttttt gactgtaaat aaaattgtaa ggagtaaaaa
     2641 gtactttttt ttctaaaaaa atgtaattaa gtaaaagtaa aagtattgat ttttaattgt
     2701 actcaagtaa agtaaaaatc cccaaaaata atacttaagt acagtaatca agtaaaatta
     2761 ctcaagtact ttacacctct ggttcttgac cccctacctt cagcaagccc agcagatcca
     2821 ctagttctag agcggccgcc accgcggtgg agctccagct tttgttccct ttagtgaggg
     2881 ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     2941 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     3001 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     3061 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     3121 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     3181 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     3241 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     3301 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     3361 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     3421 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     3481 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     3541 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     3601 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     3661 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     3721 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     3781 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     3841 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     3901 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggc
     3961 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     4021 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     4081 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     4141 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     4201 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     4261 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     4321 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     4381 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     4441 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     4501 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     4561 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     4621 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     4681 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     4741 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     4801 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     4861 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     4921 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     4981 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     5041 ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     5101 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     5161 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     5221 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     5281 gctggctaag aactcatcag cctccccggt ccatctaccc acgtaccaat gcaccaattg
     5341 gccacaatga cggctactac atggtgccat tccttcctct ttataggaat ggagactacc
     5401 tcctgtccaa caatgctctt ggatacgagt acgcctacct gttggaccca ggtcattgca
     5461 caacaccaga aatgccctct gatctgcaaa agacgtgaat atctgttcag acacccatat
     5521 ccactctgtt ccacacaggt cagaggtttg tccaggagtt cttgacagag gtgtaaaaag
     5581 tactcaaaaa ttttactcaa gtgaaagtac aagtacttag ggaaaatttt actcaattaa
     5641 aagtaaaagt atctggctag aatcttactt gagtaaaagt aaaaaagtac tccattaaaa
     5701 ttgtacttga gtattaagga agtaaaagta aaagcaagaa agaaaactag agattcttgt
     5761 ttaagctttt aatctcaaaa aacattaaat gaaatgcata caaggtttta tcctgcttta
     5821 gaactgtttg tatttaatta tcaaactata agacagacaa tctaatgcca gtacacgcta
     5881 ctcaaagttg taaaacctca gatttaactt cagtagaagc tgattctcaa aattgttagt
     5941 gtcaagccta gctcttttgg ggctgaaaag caatcctgca gtgctgaaaa gcctctcaca
     6001 ggcagccgat gcgggaagag gtgtattagt cttgatagag aggctgcaaa tagcaggaaa
     6061 cgtgagcaga gactccctgg tgtctgaaac acaggccaga tgggccctcg agcaggaaac
     6121 agctatgacc atgattacgc caagctatca actttgtata gaaaagttgg ggtaatcgct
     6181 tatcctcgga taaacaatta tcctcacggg taatcgctta tccgctcggg taatcgctta
     6241 tcctcgggta atcgcttatc cttgcaaggg tcgactctag agggtatata atggatccca
     6301 tcgcgtctca gcctcacttt gagctcctcc acacgaattc cctcgacctc gaagacgcgt
     6361 caagtttgta caaaaaagca ggctggacgg atccaccggt cgccaccatg gtgagcaagg
     6421 gcgaggagga taacatggcc tctctcccag cgacacatga gttacacatc tttggctcca
     6481 tcaacggtgt ggactttgac atggtgggtc agggcaccgg caatccaaat gatggttatg
     6541 aggagttaaa cctgaagtcc accaagggtg acctccagtt ctccccctgg attctggtcc
     6601 ctcatatcgg gtatggcttc catcagtacc tgccctaccc tgacgggatg tcgcctttcc
     6661 aggccgccat ggtagatggc tccggctacc aagtccatcg cacaatgcag tttgaagatg
     6721 gtgcctccct tactgttaac taccgctaca cctacgaggg aagccacatc aaaggagagg
     6781 cccaggtgaa ggggactggt ttccctgctg acggtcctgt gatgaccaac tcgctgaccg
     6841 ctgcggactg gtgcaggtcg aagaagactt accccaacga caaaaccatc atcagtacct
     6901 ttaagtggag ttacaccact ggaaatggca agcgctaccg gagcactgcg cggaccacct
     6961 acacctttgc caagccaatg gcggctaact atctgaagaa ccagccgatg tacgtgttcc
     7021 gtaagacgga gctcaagcac tccaagaccg agctcaactt caaggagtgg caaaaggcct
     7081 ttaccgatgt gatgggcatg gacgagctgt acaaggagct gcagaacaag ctggaagagt
     7141 tggatctgga ctctcgagac ccagctttct tgtacaaagt ggggggatcc ggagccacga
     7201 acttctctct gttaaagcaa gcaggagacg tggaagaaaa ccccggtcct atggtgtctg
     7261 agctgattaa ggagaacatg cacatgaagc tgtacatgga gggcaccgtg aacaaccacc
     7321 acttcaagtg cacatccgag ggcgaaggca agccctacga gggcacccag accatgagaa
     7381 tcaaggccgt cgagggcggc cctctcccct tcgccttcga catcctggct accagcttca
     7441 tgtacggcag caaaaccttc atcaaccaca cccagggcat ccccgacttc tttaagcagt
     7501 ccttccctga gggcttcaca tgggagagag tcaccacata cgaagacggg ggcgtgctga
     7561 ccgctaccca ggacaccagc ctccaggacg gctgcctcat ctacaacgtc aagatcagag
     7621 gggtgaactt cccatccaac ggccctgtga tgcagaagaa aacactcggc tgggaggcct
     7681 ccaccgagac cctgtacccc gctgacggcg gcctggaagg cagagccgac atggccctga
     7741 agctcgtggg cgggggccac ctgatctgca acttgaagac cacatacaga tccaagaaac
     7801 ccgctaagaa cctcaagatg cccggcgtct actatgtgga cagaagactg gaaagaatca
     7861 aggaggccga caaagagacc tacgtcgagc agcacgaggt ggctgtggcc agatactgcg
     7921 acctccctag caaactgggg cacagaggcg gtggaagatc tcgagctcaa gcttcgaatt
     7981 cccgggtcga tccaaaaaag aagagaaagg tagatccaaa aaagaagaga aaggtagatc
     8041 caaaaaagaa gagaaaggta taggtaccgc gggcccggga tccaccggat ctagaactat
     8101 agtgagtcgt attacgtaga tccagacatg ataagataca ttgatgagtt tggacaaacc
     8161 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     8221 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
     8281 tttcaggttc agggggaggt gtgggaggtt ttttccaact tta
//
