LOCUS       sequence-181093-        7589 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Human KRasG12V 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 7589)
  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 7589)
  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-KrasG12V
FEATURES             Location/Qualifiers
     source          1..7589
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(677..696)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(678..696)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(706..723)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(706..722)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(715..737)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      864..1319
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(951..970)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1161..1182
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1345..1449
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1450..1518,1519..2310)
                     /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(1668..1687)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2481..3069
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2970..2989
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3223..3240
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3357..3378
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3393..3398,3399..3416,3417..3423)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3431..3447
                     /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     3436..3458
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3455..3471
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3455..3471
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3490..3510
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3492..3510
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3918..3937)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    3947..4028
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     3972..3991
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(4154..4684,4685..4687,4688..4720))
                     /codon_start=1
                     /product="human oncoprotein generated by the G12V mutation 
                     in the small GTPase K-Ras"
                     /label=K-Ras (G12V)
                     /translation="MTEYKLVVVGAVGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVK
                     DSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREI
                     RKHKEKMSKDGKKKKKKSKTKCVIM"
     CDS             complement(4745..5452)
                     /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(5020..5039)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     5289..5307
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      5449..5458
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5525..5571)
                     /label=minimal CaMV 35S promoter
                     /note="minimal 35S promoter from cauliflower mosaic virus"
     polyA_signal    6182..6406
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     6395..6412
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(join(6435..7148,7149..7151,7152..7154))
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6480..6501)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     6829..6848
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7088..7109
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      7151..7160
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    7313..7346
                     /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)."
ORIGIN
        1 ctatgctata ggctgtatac ctatggctaa gagaggtctg tgttcaggtc agaggtcacg
       61 tgatgataat ccgagcagag gagtcgctcg ctgatacact ccagagcaga gtagatttca
      121 tactgaagtt aataatgtag catgacatat attagatggt gtgtgtgtgt gtgtgtatat
      181 acatatatat atatatatgg tagcgtattt agatggaatt tattgatgta ttatttatta
      241 tatgtatata cagatgaagt caaaattttt cgctccctgt atattttccc cttaaatcct
      301 gtttaagaga aagaaacata tttctaatca taatagtttt aataactcat ttctaataac
      361 tgatttcttc tctctttgtc atgatgatag caccaattgc tcgagggatc tgttgcagtc
      421 atcccgaatt agaaaatacg gtaacgaaac gggatcatcc cgattaaaaa cgggatcccg
      481 gtgaaacggt cgggaaacta gctctaccgt ttccgtttcc gtttaccgtt ttgtatatcc
      541 cgtttccgtt ccgttttcgt tttttacctc gggttcgaaa tcgatcggga taaaactaac
      601 aaaatcggtt atacgataac ggtcggtacg ggattttccc atcctacttt catccctgcg
      661 tcgggtaccc aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt
      721 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
      781 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
      841 gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
      901 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
      961 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     1021 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     1081 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     1141 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     1201 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     1261 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
     1321 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     1381 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     1441 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     1501 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     1561 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     1621 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     1681 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     1741 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     1801 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     1861 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     1921 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     1981 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     2041 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     2101 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     2161 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     2221 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     2281 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     2341 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     2401 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     2461 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     2521 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     2581 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     2641 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     2701 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     2761 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     2821 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     2881 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     2941 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     3001 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     3061 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     3121 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     3181 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     3241 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     3301 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     3361 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
     3421 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac
     3481 gccaagcgcg caattaaccc tcactaaagg gaacaaaagc tggagctccc cgactaggga
     3541 tgaaaacggt cggtaacggt cggtaaaata cctctaccgt tttcattttc atatttaact
     3601 tgcgggacgg aaacgaaaac gggatatacc ggtaacgaaa acgaacggga taaatacggt
     3661 aatcgaaaac cgatacgatc cggtcgggtt aaagtcgaaa tcggacggga accggtattt
     3721 ttgttcggta aaatcacaca tgaaaacata tattcaaaac ttaaaaacaa atataaaaaa
     3781 ttgtaaacac aagtcttaat taaacataga taaaatccat ataaatctgg agcacacata
     3841 gtttaatgta gcacataagt gataagtctt gggctcttgg ctaacataag aagccatata
     3901 agtctactct acgtattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat
     3961 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
     4021 aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg
     4081 aggtttttta aagcaagtaa aacctctaca aatgtggtat ggctgattat gatctagagt
     4141 cgcggccgct ttattacata attacacact ttgtctttga cttctttttc ttctttttac
     4201 catctttgct catcttttct ttatgttttc gaatttctcg aactaatgta tagaaggcat
     4261 catcaacacc ctgtcttgtc tttgctgatg tttcaataaa aggaattcca taacttcttg
     4321 ctaagtcctg agcctgtttt gtgtctactg ttctggaagg caaatcacat ttatttccta
     4381 ctaggaccat aggtacatct tcagagtcct taactctttt aatttgttct ctataatggt
     4441 gaatatcttc aaatgattta gtattattta tggcaaatac acaaagaaag ccctccccag
     4501 tcctcatgta ctggtccctc attgcactgt actcctcttg acctgctgtg tcgagaatat
     4561 ccaagagaca ggtttctcca tcaattacta cttgcttcct gtaggaatcc tctattgttg
     4621 gatcatattc gtccacaaaa tgattctgaa ttagctgtat cgtcaaggca ctcttgccta
     4681 cgccaacagc tccaactacc acaagtttat attcagtcat accagaacca cctcgcgagt
     4741 cgaccttgta cagctcgtcc atgccgccgg tggagtggcg gccctcggcg cgttcgtact
     4801 gttccacgat ggtgtagtcc tcgttgtggg aggtgatgtc caacttgatg ttgacgttgt
     4861 aggcgccggg cagctgcacg ggcttcttgg ccttgtaggt ggtcttgacc tcagcgtcgt
     4921 agtggccgcc gtccttcagc ttcagcctct gcttgatctc gcccttcagg gcgccgtcct
     4981 cggggtacat ccgctcggag gaggcctccc agcccatggt cttcttctgc attacggggc
     5041 cgtcggaggg gaagttggtg ccgcgcagct tcaccttgta gatgaactcg ccgtcctgca
     5101 gggaggagtc ctgggtcacg gtcaccacgc cgccgtcctc gaagttcatc acgcgctccc
     5161 acttgaagcc ctcggggaag gacagcttca agtagtcggg gatgtcggcg gggtgcttca
     5221 cgtaggcctt ggagccgtac atgaactgag gggacaggat gtcccaggcg aagggcaggg
     5281 ggccaccctt ggtcaccttc agcttggcgg tctgggtgcc ctcgtagggg cggccctcgc
     5341 cctcgccctc gatctcgaac tcgtggccgt tcacggagcc ctccatgtgc accttgaagc
     5401 gcatgaactc cttgatgatg gccatgttat cctcctcgcc cttgctcacc atggtggcgg
     5461 gatcccgggc ccgcggtacc gtcgactgca gaattcgagg ctagagtcga ctagcttcag
     5521 cgtgtcctct ccaaatgaaa tgaacttcct tatatagagg aagggtcttg cgaagatcct
     5581 agagtcgagc atattacatg gcattaccac catatacata tccatataca tatccatatc
     5641 taatcttacc tcgactgctg tatataaaac cagtggttat atgtacagta ctgctgtata
     5701 taaaaccagt ggttatatgt acagtacgtc gactgctgta tataaaacca gtggttatat
     5761 gtacagtact gctgtatata aaaccagtgg ttatatgtac agtacgtcga ggggatgata
     5821 atgcgattag ttttttagcc tcgacctgca gcccaagcgg catgcaagct gtcgaggggg
     5881 gatcaattcc cgatctagta acatagatga caccgcgcgc gataatttat cctagtttgc
     5941 gcgctatatt ttgttttcta tcgcgtatta aatgtataat tgcgggactc taatcataaa
     6001 aacccatctc ataaataacg tcatgcatta catgttaatt attacatgct taacgtaatt
     6061 caacagaaat tatatgataa tcatcgcaag accggcaaca ggattcaatc ttaatttaaa
     6121 ttataactag tcgacgatcg gcggcctagg cgccacgtgc tggttctttc cgcctcagaa
     6181 gccatagagc ccaccgcatc cccagcatgc ctgctattgt cttcccaatc ctcccccttg
     6241 ctgtcctgcc ccaccccacc ccccagaata gaatgacacc tactcagaca atgcgatgca
     6301 atttcctcat tttattagga aaggacagtg ggagtggcac cttccagggt caaggaaggc
     6361 acgggggagg ggcaaacaac agatggctgg caactagaag gcacagtcga ggctgatcag
     6421 cgggtttggc cgctttactt gtacagctcg tccatgccga gagtgatccc ggcggcggtc
     6481 acgaactcca gcaggaccat gtgatcgcgc ttctcgttgg ggtctttgct cagggcggac
     6541 tgggtgctca ggtagtggtt gtcgggcagc agcacggggc cgtcgccgat gggggtgttc
     6601 tgctggtagt ggtcggcgag ctgcacgctg ccgtcctcga tgttgtggcg gatcttgaag
     6661 ttggccttga tgccgttctt ctgcttgtcg gcggtgatat agacgttgtg gctgatgtag
     6721 ttgtactcca gcttgtgccc caggatgttg ccgtcctcct tgaagtcgat gcccttcagc
     6781 tcgatgcggt tcaccagggt gtcgccctcg aacttcacct cggcgcgggt cttgtagttg
     6841 ccgtcgtcct tgaagaagat ggtgcgctcc tggacgtagc cttcgggcat ggcggacttg
     6901 aagaagtcgt gctgcttcat gtggtcgggg tagcggctga agcactgcac gccccaggtc
     6961 agggtggtca cgagggtggg ccagggcacg ggcagcttgc cggtggtgca gatgaacttc
     7021 agggtcagct tgccgtaggt ggcatcgccc tcgccctcgc cggacacgct gaacttgtgg
     7081 ccgtttacgt cgccgtccag ctcgaccagg atgggcacca ccccggtgaa cagctcctcg
     7141 cccttgctca ccatggtggc gggatcccct agcaaaaata cagatactgc agtttctgat
     7201 aggaaggttg agcagaatag caggggaagg aaacaggagg aagacactta cctgttaatt
     7261 aacacgggga atgcgcgttc caatgcaccg ttcccggcaa cctagcatgc agataacttc
     7321 gtataaagta tcctatacga agttatggca acgcgtttaa actacagctg tgctgggaag
     7381 gctggctcgc tctggccttc ctcaccactg ccgcagcttt tatacgctgg caagggcagc
     7441 acagcggcgc gcggcggaca atagaaacac gccgtcatca gagttcagcc gtgccgaagc
     7501 gcagggtcat cgcatcgccc agaaggccgt aagcatttta ctgtcaatgg aaagccctga
     7561 cggcggcact ttccctctca gcccgagct
//
