LOCUS       sequence-181092-        7592 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Human HRasG12V fused to mCherry downstream of LexOP, placed within a
            non-autonomous maize dissociation (Ds) element for integration in 
            zebrafish. Contains pCrysB-ECFP for screening..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7592)
  AUTHORS   Kenyon A, Gavriouchkina D, Zorman J, Chong-Morrison V, Napolitani G,
            Cerundolo V, Sauka-Spengler T
  TITLE     Generation of a double binary transgenic zebrafish model to study 
            myeloid gene regulation in response to oncogene activation in 
            melanocytes.
  JOURNAL   Dis Model Mech. 2018 Apr 6;11(4). pii: dmm.030056. doi: 
            10.1242/dmm.030056.
  PUBMED    29666124
REFERENCE   2  (bases 1 to 7592)
  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"; journalName: "Dis Model 
            Mech."; date: "2018-04-6"; volume: "11(4). pii: dmm.030056. doi"; 
            pages: " 10.1242/dmm.030056"
COMMENT     Sequence Label: pCrys-beta:ECFP-LexOP:mCherry-HrasG12V
FEATURES             Location/Qualifiers
     source          1..7592
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     165..184
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     424..445
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      487..496
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    649..682
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     primer_bind     complement(1602..1621)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1603..1621)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1631..1648)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1631..1647)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1640..1662)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1789..2244
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1876..1895)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2086..2107
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2270..2374
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2375..2443,2444..3235)
                     /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(2593..2612)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3406..3994
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3895..3914
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4148..4165
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4282..4303
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4318..4323,4324..4341,4342..4348)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4356..4372
                     /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     4361..4383
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4380..4396
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4380..4396
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4415..4435
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4417..4435
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4843..4862)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    4872..4953
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     4897..4916
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(5079..5612,5613..5615,5616..5648))
                     /codon_start=1
                     /product="human oncoprotein generated by the G12V mutation 
                     in the small GTPase H-Ras"
                     /label=H-Ras (G12V)
                     /translation="MTEYKLVVVGAVGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHQYREQIKRVK
                     DSDDVPMVLVGNKCDLAARTVESRQAQDLARSYGIPYIETSAKTRQGVEDAFYTLVREI
                     RQHKLRKLNPPDESGPGCMSCKCVLS"
     CDS             complement(5673..6380)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(5948..5967)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     6217..6235
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      6377..6386
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6453..6499)
                     /label=minimal CaMV 35S promoter
                     /note="minimal 35S promoter from cauliflower mosaic virus"
     polyA_signal    7110..7334
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     7323..7340
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(join(7363..7592,1..484,485..487,488..490))
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(7408..7429)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 ccttgatgcc gttcttctgc ttgtcggcgg tgatatagac gttgtggctg atgtagttgt
       61 actccagctt gtgccccagg atgttgccgt cctccttgaa gtcgatgccc ttcagctcga
      121 tgcggttcac cagggtgtcg ccctcgaact tcacctcggc gcgggtcttg tagttgccgt
      181 cgtccttgaa gaagatggtg cgctcctgga cgtagccttc gggcatggcg gacttgaaga
      241 agtcgtgctg cttcatgtgg tcggggtagc ggctgaagca ctgcacgccc caggtcaggg
      301 tggtcacgag ggtgggccag ggcacgggca gcttgccggt ggtgcagatg aacttcaggg
      361 tcagcttgcc gtaggtggca tcgccctcgc cctcgccgga cacgctgaac ttgtggccgt
      421 ttacgtcgcc gtccagctcg accaggatgg gcaccacccc ggtgaacagc tcctcgccct
      481 tgctcaccat ggtggcggga tcccctagca aaaatacaga tactgcagtt tctgatagga
      541 aggttgagca gaatagcagg ggaaggaaac aggaggaaga cacttacctg ttaattaaca
      601 cggggaatgc gcgttccaat gcaccgttcc cggcaaccta gcatgcagat aacttcgtat
      661 aaagtatcct atacgaagtt atggcaacgc gtttaaacta cagctgtgct gggaaggctg
      721 gctcgctctg gccttcctca ccactgccgc agcttttata cgctggcaag ggcagcacag
      781 cggcgcgcgg cggacaatag aaacacgccg tcatcagagt tcagccgtgc cgaagcgcag
      841 ggtcatcgca tcgcccagaa ggccgtaagc attttactgt caatggaaag ccctgacggc
      901 ggcactttcc ctctcagccc gagctctatg ctataggctg tatacctatg gctaagagag
      961 gtctgtgttc aggtcagagg tcacgtgatg ataatccgag cagaggagtc gctcgctgat
     1021 acactccaga gcagagtaga tttcatactg aagttaataa tgtagcatga catatattag
     1081 atggtgtgtg tgtgtgtgtg tatatacata tatatatata tatggtagcg tatttagatg
     1141 gaatttattg atgtattatt tattatatgt atatacagat gaagtcaaaa tttttcgctc
     1201 cctgtatatt ttccccttaa atcctgttta agagaaagaa acatatttct aatcataata
     1261 gttttaataa ctcatttcta ataactgatt tcttctctct ttgtcatgat gatagcacca
     1321 attgctcgag ggatctgttg cagtcatccc gaattagaaa atacggtaac gaaacgggat
     1381 catcccgatt aaaaacggga tcccggtgaa acggtcggga aactagctct accgtttccg
     1441 tttccgttta ccgttttgta tatcccgttt ccgttccgtt ttcgtttttt acctcgggtt
     1501 cgaaatcgat cgggataaaa ctaacaaaat cggttatacg ataacggtcg gtacgggatt
     1561 ttcccatcct actttcatcc ctgcgtcggg tacccaattc gccctatagt gagtcgtatt
     1621 acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
     1681 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
     1741 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta
     1801 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     1861 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     1921 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     1981 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     2041 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     2101 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     2161 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     2221 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     2281 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     2341 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     2401 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     2461 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     2521 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     2581 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     2641 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     2701 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     2761 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     2821 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     2881 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     2941 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     3001 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     3061 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     3121 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     3181 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     3241 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     3301 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     3361 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     3421 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     3481 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     3541 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     3601 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     3661 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     3721 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     3781 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3841 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3901 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3961 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     4021 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     4081 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     4141 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
     4201 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     4261 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
     4321 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
     4381 aggaaacagc tatgaccatg attacgccaa gcgcgcaatt aaccctcact aaagggaaca
     4441 aaagctggag ctccccgact agggatgaaa acggtcggta acggtcggta aaatacctct
     4501 accgttttca ttttcatatt taacttgcgg gacggaaacg aaaacgggat ataccggtaa
     4561 cgaaaacgaa cgggataaat acggtaatcg aaaaccgata cgatccggtc gggttaaagt
     4621 cgaaatcgga cgggaaccgg tatttttgtt cggtaaaatc acacatgaaa acatatattc
     4681 aaaacttaaa aacaaatata aaaaattgta aacacaagtc ttaattaaac atagataaaa
     4741 tccatataaa tctggagcac acatagttta atgtagcaca taagtgataa gtcttgggct
     4801 cttggctaac ataagaagcc atataagtct actctacgta ttgatgagtt tggacaaacc
     4861 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     4921 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
     4981 tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt
     5041 ggtatggctg attatgatct agagtcgcgg ccgctttatc aggagagcac acacttgcag
     5101 ctcatgcagc cggggccact ctcatcagga gggttcagct tccgcagctt gtgctgccgg
     5161 atctcacgca ccaacgtgta gaaggcatcc tccactccct gccgggtctt ggccgaggtc
     5221 tcgatgtagg ggatgccgta gcttcgggcg aggtcctgag cctgccgaga ttccacagtg
     5281 cgtgcagcca ggtcacactt gttccccacc agcaccatgg gcacgtcatc cgagtccttc
     5341 acccgtttga tctgctccct gtactggtgg atgtcctcaa aagacttggt gttgttgatg
     5401 gcaaacacac acaggaagcc ctccccggtg cgcatgtact ggtcccgcat ggcgctgtac
     5461 tcctcctggc cggcggtatc caggatgtcc aacaggcacg tctccccatc aatgaccacc
     5521 tgcttccggt aggaatcctc tatagtgggg tcgtattcgt ccacaaaatg gttctggatc
     5581 agctggatgg tcagcgcact cttgcccaca ccgacggcgc ccaccaccac cagcttatat
     5641 tccgtcatac cagaaccacc tcgcgagtcg accttgtaca gctcgtccat gccgccggtg
     5701 gagtggcggc cctcggcgcg ttcgtactgt tccacgatgg tgtagtcctc gttgtgggag
     5761 gtgatgtcca acttgatgtt gacgttgtag gcgccgggca gctgcacggg cttcttggcc
     5821 ttgtaggtgg tcttgacctc agcgtcgtag tggccgccgt ccttcagctt cagcctctgc
     5881 ttgatctcgc ccttcagggc gccgtcctcg gggtacatcc gctcggagga ggcctcccag
     5941 cccatggtct tcttctgcat tacggggccg tcggagggga agttggtgcc gcgcagcttc
     6001 accttgtaga tgaactcgcc gtcctgcagg gaggagtcct gggtcacggt caccacgccg
     6061 ccgtcctcga agttcatcac gcgctcccac ttgaagccct cggggaagga cagcttcaag
     6121 tagtcgggga tgtcggcggg gtgcttcacg taggccttgg agccgtacat gaactgaggg
     6181 gacaggatgt cccaggcgaa gggcaggggg ccacccttgg tcaccttcag cttggcggtc
     6241 tgggtgccct cgtaggggcg gccctcgccc tcgccctcga tctcgaactc gtggccgttc
     6301 acggagccct ccatgtgcac cttgaagcgc atgaactcct tgatgatggc catgttatcc
     6361 tcctcgccct tgctcaccat ggtggcggga tcccgggccc gcggtaccgt cgactgcaga
     6421 attcgaggct agagtcgact agcttcagcg tgtcctctcc aaatgaaatg aacttcctta
     6481 tatagaggaa gggtcttgcg aagatcctag agtcgagcat attacatggc attaccacca
     6541 tatacatatc catatacata tccatatcta atcttacctc gactgctgta tataaaacca
     6601 gtggttatat gtacagtact gctgtatata aaaccagtgg ttatatgtac agtacgtcga
     6661 ctgctgtata taaaaccagt ggttatatgt acagtactgc tgtatataaa accagtggtt
     6721 atatgtacag tacgtcgagg ggatgataat gcgattagtt ttttagcctc gacctgcagc
     6781 ccaagcggca tgcaagctgt cgagggggga tcaattcccg atctagtaac atagatgaca
     6841 ccgcgcgcga taatttatcc tagtttgcgc gctatatttt gttttctatc gcgtattaaa
     6901 tgtataattg cgggactcta atcataaaaa cccatctcat aaataacgtc atgcattaca
     6961 tgttaattat tacatgctta acgtaattca acagaaatta tatgataatc atcgcaagac
     7021 cggcaacagg attcaatctt aatttaaatt ataactagtc gacgatcggc ggcctaggcg
     7081 ccacgtgctg gttctttccg cctcagaagc catagagccc accgcatccc cagcatgcct
     7141 gctattgtct tcccaatcct cccccttgct gtcctgcccc accccacccc ccagaataga
     7201 atgacaccta ctcagacaat gcgatgcaat ttcctcattt tattaggaaa ggacagtggg
     7261 agtggcacct tccagggtca aggaaggcac gggggagggg caaacaacag atggctggca
     7321 actagaaggc acagtcgagg ctgatcagcg ggtttggccg ctttacttgt acagctcgtc
     7381 catgccgaga gtgatcccgg cggcggtcac gaactccagc aggaccatgt gatcgcgctt
     7441 ctcgttgggg tctttgctca gggcggactg ggtgctcagg tagtggttgt cgggcagcag
     7501 cacggggccg tcgccgatgg gggtgttctg ctggtagtgg tcggcgagct gcacgctgcc
     7561 gtcctcgatg ttgtggcgga tcttgaagtt gg
//
