LOCUS       sequence-374477-        6143 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mOrange2-KRAS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6143)
  AUTHORS   Shi Z, Graber ZT, Baumgart T, Stone HA, Cohen AE
  TITLE     Cell Membranes Resist Flow.
  JOURNAL   Cell. 2018 Dec 13;175(7):1769-1779.e13. doi: 
            10.1016/j.cell.2018.09.054. Epub 2018 Nov 1.
  PUBMED    30392960
REFERENCE   2  (bases 1 to 6143)
  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"; doi: 
            "10.1016/j.cell.2018.09.054"; journalName: "Cell"; date: 
            "2018-12-13- 13"; volume: "175"; issue: "7"; pages: "1769-1779.e13"
COMMENT     Sequence Label: ZS041:mOrange2-KRAS
FEATURES             Location/Qualifiers
     source          1..6143
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(66..82)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(66..82)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(79..101)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    90..106
                     /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(114..120,121..138,139..144))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    159..180
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(297..314)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(468..1056)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(548..567)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1227..2018,2019..2087))
                     /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     1850..1869
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2088..2192)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2267..2286)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2458..2837
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2838..3041
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2992..3012
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3086..3105
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3086..3104
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      3118..3127
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3124..3831
                     /codon_start=1
                     /product="photostable monomeric orange derivative of DsRed 
                     fluorescent protein (Shaner et al., 2008)"
                     /label=mOrange2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEENNMAIIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEG
                     FQTAKLKVTKGGPLPFAWDILSPHFTYGSKAYVKHPADIPDYFKLSFPEGFKWERVMNY
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GKIKMRLKLKDGGHYTSEVKTTYKAKKPVQLPGAYIVDIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(3957..3974)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3963..4187
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4233..4661
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4320..4339)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4530..4551
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4670..4690)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4675..5004
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4855..4990
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4917..4936
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5071..5865
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5125..5144)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5735..5754
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    join(6039..6143,1..17)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6076..6095)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     join(6130..6143,1..6)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa
       61 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
      121 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
      181 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
      241 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
      301 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
      361 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
      421 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
      481 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
      541 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
      601 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
      661 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
      721 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
      781 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
      841 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
      901 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
      961 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     1021 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     1081 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     1141 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     1201 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     1261 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     1321 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     1381 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     1441 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     1501 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     1561 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     1621 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     1681 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     1741 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     1801 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     1861 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     1921 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     1981 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     2041 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     2101 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     2161 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     2221 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
     2281 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
     2341 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
     2401 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
     2461 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
     2521 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
     2581 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
     2641 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
     2701 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
     2761 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
     2821 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
     2881 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
     2941 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
     3001 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tctctggcta actagagaac
     3061 ccactgctta ctggcttatc gaaattaata cgactcacta tagggagacc caagcttgcc
     3121 accatggtga gcaagggcga ggagaataac atggccatca tcaaggagtt catgcgcttc
     3181 aaggtgcgca tggagggctc cgtgaacggc cacgagttcg agatcgaggg cgagggcgag
     3241 ggccgcccct acgagggctt tcagaccgct aagctgaagg tgaccaaggg tggccccctg
     3301 cccttcgcct gggacatcct gtcccctcat ttcacctacg gctccaaggc ctacgtgaag
     3361 caccccgccg acatccccga ctacttcaag ctgtccttcc ccgagggctt caagtgggag
     3421 cgcgtgatga actacgagga cggcggcgtg gtgaccgtga cccaggactc ctccctgcag
     3481 gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc cgacggcccc
     3541 gtgatgcaga agaagaccat gggctgggag gcctcctccg agcggatgta ccccgaggac
     3601 ggtgccctga agggcaagat caagatgagg ctgaagctga aggacggcgg ccactacacc
     3661 tccgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg cgcctacatc
     3721 gtcgacatca agttggacat cacctcccac aacgaggact acaccatcgt ggaacagtac
     3781 gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa gaccggtggt
     3841 aaaaagaaga aaaagaagtc aaagacaaag tgtgtaatta tgtaatgaat tctgcagata
     3901 tccatcacag tggcggccgc tcgagtctag agggcccgtt taaacccgct gatcagcctc
     3961 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     4021 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     4081 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     4141 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     4201 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     4261 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     4321 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     4381 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     4441 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     4501 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     4561 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     4621 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     4681 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     4741 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     4801 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     4861 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     4921 tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt
     4981 tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct
     5041 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt
     5101 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     5161 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     5221 accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg
     5281 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     5341 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     5401 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     5461 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     5521 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     5581 ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     5641 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     5701 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     5761 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     5821 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     5881 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     5941 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     6001 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     6061 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     6121 attctagttg tggtttgtcc aaa
//
