LOCUS       sequence-333693-        7524 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses FLAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7524)
  AUTHORS   Yu B, Swatkoski S, Holly A, Lee LC, Giroux V, Lee CS, Hsu D, Smith 
            JL, Yuen G, Yue J, Ann DK, Simpson RM, Creighton CJ, Figg WD, Gucek 
            M, Luo J
  TITLE     Oncogenesis driven by the Ras/Raf pathway requires the SUMO E2 
            ligase Ubc9.
  JOURNAL   Proc Natl Acad Sci U S A. 2015 Apr 7;112(14):E1724-33. doi: 
            10.1073/pnas.1415569112. Epub 2015 Mar 24.
  PUBMED    25805818
REFERENCE   2  (bases 1 to 7524)
  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: 
            "10.1073/pnas.1415569112"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2015-04-7- 7"; volume: "112"; issue: "14"; pages: "E1724-33"
COMMENT     Sequence Label: MSCVhyg-HA-FLAG_Ubc9_C93A
FEATURES             Location/Qualifiers
     source          1..7524
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    579..920
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             987..1403
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1333..1355
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1371..1387
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     regulatory      1416..1425
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1425..1448
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1461..1487
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    1503..1527
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        2017..2516
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2052..2075)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2449..2468
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2534..3559
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             2534..3559
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     LTR             3917..4431
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4593..4609
                     /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(4617..4623,4624..4641,4642..4647))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4662..4683
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4800..4817)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4971..5559)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5051..5070)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5730..6521,6522..6590))
                     /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     6353..6372
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6591..6695)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6763..6781
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6819..6841)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6941..6960
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7154..7176
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7304..7323
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(7523..7524,1..515)
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
ORIGIN
        1 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat
       61 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca
      121 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
      181 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt
      241 ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc
      301 gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc
      361 ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt attcccaata
      421 aagcctcttg ctgtttgcat ccgaatcgtg gactcgctga tccttgggag ggtctcctca
      481 gattgattga ctgcccacct cgggggtctt tcatttggag gttccaccga gatttggaga
      541 cccctgccca gggaccaccg acccccccgc cgggaggtaa gctggccagc ggtcgtttcg
      601 tgtctgtctc tgtctttgtg cgtgtttgtg ccggcatcta atgtttgcgc ctgcgtctgt
      661 actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac gagttctgaa
      721 cacccggccg caaccctggg agacgtccca gggactttgg gggccgtttt tgtggcccga
      781 cctgaggaag ggagtcgatg tggaatccga ccccgtcagg atatgtggtt ctggtaggag
      841 acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt tggaaccgaa
      901 gccgcgcgtc ttgtctgctg cagcgctgca gcatcgttct gtgttgtctc tgtctgactg
      961 tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta agtttgacct
     1021 taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga
     1081 gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag
     1141 acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc
     1201 cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc
     1261 cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg
     1321 ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag
     1381 ccctcactcc ttctctaggc gccggaatta gatctgccac catggactac aaggatgacg
     1441 atgacaagct cgatggagga tacccatacg atgttccaga ttacgctgct cgaggttatc
     1501 aaacaagttt gtacaaaaaa gcaggcttaa tgtcagggat cgccctcagc agactcgccc
     1561 aggagaggaa agcatggagg aaagaccacc catttggttt cgtggctgtt ccaacaaaaa
     1621 atcccgatgg cacgatgaac ctcatgaact gggagtgcgc cattccggga aagaaaggga
     1681 ctccgtggga aggaggcttg tttaaactac ggatgctttt caaagatgat tatccatctt
     1741 cgccaccaaa atgtaaattc gaaccaccat tatttcaccc gaatgtgtac ccttcgggga
     1801 cagtggccct gtccatctta gaggaggaca aggactggag gccagccatc acaatcaaac
     1861 agatcctatt aggaatacag gaacttctaa atgaaccaaa tatccaagac ccagctcaag
     1921 cagaggccta cacgatttac tgccaaaaca gagtggagta cgagaaaagg gtccgagcac
     1981 aagccaagaa gtttgcgccc tcataagaat tctaccgggt aggggaggcg cttttcccaa
     2041 ggcagtctgg agcatgcgct ttagcagccc cgctgggcac ttggcgctac acaagtggcc
     2101 tctggcctcg cacacattcc acatccaccg gtaggcgcca accggctccg ttctttggtg
     2161 gccccttcgc gccaccttct actcctcccc tagtcaggaa gttccccccc gccccgcagc
     2221 tcgcgtcgtg caggacgtga caaatggaag tagcacgtct cactagtctc gtgcagatgg
     2281 acagcaccgc tgagcaatgg aagcgggtag gcctttgggg cagcggccaa tagcagcttt
     2341 gctccttcgc tttctgggct cagaggctgg gaaggggtgg gtccgggggc gggctcaggg
     2401 gcgggctcag gggcggggcg ggcgcccgaa ggtcctccgg aggcccggca ttctgcacgc
     2461 ttcaaaagcg cacgtctgcc gcgctgttct cctcttcctc atctccgggc ctttcgacct
     2521 gcatcccgcc accatgaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat
     2581 cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc
     2641 tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg
     2701 tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga
     2761 agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca
     2821 gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc
     2881 ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt
     2941 cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga
     3001 tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca
     3061 ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca
     3121 cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga
     3181 ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag
     3241 gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct
     3301 tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca
     3361 gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat
     3421 cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt
     3481 ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg
     3541 tccgagggca aaggaataga gtagatgccg accgaacaag agctgatttc gagaacgcct
     3601 cagccagcaa ctcgcgcgag cctagcaagg caaatgcgag agaacggcct tacgcttggt
     3661 ggcacagttc tcgtccacag ttcgctaagc tcgctcggct gggtcgcggg agggccggtc
     3721 gcagtgattc aggcccttct ggattgtgtt ggtccccagg gcacgattgt catgcccacg
     3781 cactcgggtg atctgactga tcccgcagat tggagatcgc cgcccgtgcc tgccgattgg
     3841 gtgcagatcc gtcgacctgc agccaagctt atcgataaaa taaaagattt tatttagtct
     3901 ccagaaaaag gggggaatga aagaccccac ctgtaggttt ggcaagctag cttaagtaac
     3961 gccattttgc aaggcatgga aaatacataa ctgagaatag agaagttcag atcaaggtta
     4021 ggaacagaga gacagcagaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc
     4081 ccggctcagg gccaagaaca gatggtcccc agatgcggtc ccgccctcag cagtttctag
     4141 agaaccatca gatgtttcca gggtgcccca aggacctgaa atgaccctgt gccttatttg
     4201 aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat
     4261 aaaagagccc acaacccctc actcggcgcg ccagtcctcc gatagactgc gtcgcccggg
     4321 tacccgtgta tccaataaac cctcttgcag ttgcatccga cttgtggtct cgctgttcct
     4381 tgggagggtc tcctctgagt gattgactac ccgtcagcgg gggtctttca tgggtaacag
     4441 tttcttgaag ttggagaaca acattctgag ggtaggagtc gaatattaag taatcctgac
     4501 tcaattagcc actgttttga atccacatac tccaatactc ctgaaatagt tcattatgga
     4561 cagcgcagaa agagctgggg agaattgtga aattgttatc cgctcacaat tccacacaac
     4621 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     4681 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     4741 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     4801 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     4861 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     4921 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     4981 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     5041 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     5101 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     5161 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     5221 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     5281 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     5341 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     5401 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     5461 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     5521 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     5581 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     5641 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     5701 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     5761 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     5821 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     5881 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     5941 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     6001 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     6061 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     6121 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     6181 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     6241 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     6301 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     6361 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     6421 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     6481 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     6541 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     6601 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     6661 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     6721 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     6781 ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
     6841 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
     6901 tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
     6961 ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
     7021 atcaggcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
     7081 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
     7141 acgccagggt tttcccagtc acgacgttgt aaaacgacgg cgcaaggaat ggtgcatgca
     7201 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     7261 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
     7321 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
     7381 ggcgattagt ccaatttgtt aaagacagga tatcagtggt ccaggctcta gttttgactc
     7441 aacaatatca ccagctgaag cctatagagt acgagccata gataaaataa aagattttat
     7501 ttagtctcca gaaaaagggg ggaa
//
