LOCUS       sequence-224235-        8090 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8090)
  AUTHORS   Fiordalisi JJ, Johnson RL 2nd, Ulku AS, Der CJ, Cox AD
  TITLE     Mammalian expression vectors for Ras family proteins: generation and
            use of expression constructs to analyze Ras family function.
  JOURNAL   Methods Enzymol. 2001;332:3-36. doi: 10.1016/s0076-6879(01)32189-4.
  PUBMED    11305105
REFERENCE   2  (bases 1 to 8090)
  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: "Methods 
            Enzymol."; date: "2001"; volume: "332:3-36. doi"; pages: " 
            10.1016/s0076-6879(01)32189-4"
COMMENT     Sequence Label: pCGN N-Ras wt
FEATURES             Location/Qualifiers
     source          1..8090
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     505..529
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     5'UTR           598..637
                     /label=HSV TK 5'-UTR
                     /note="5' untranslated region from the herpes simplex virus
                     thymidine kinase gene"
     CDS             667..693
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             673..699
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     intron          1325..1897
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     primer_bind     complement(2029..2048)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2094..2149
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2148..2167)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(2518..2538)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2523..2856
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2703..2842
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      2864..2873
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2870..3895
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             2870..3895
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     rep_origin      4967..5102
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5208..5225)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5379..5967)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5459..5478)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6138..6929,6930..6998))
                     /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     6761..6780
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6999..7103)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7171..7189
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7227..7249)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7349..7368
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(7435..7890)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7572..7593)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7784..7803
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8016..8038
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8030..8047
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8031..8047
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8054..8073
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        8054..8072
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagacg
      541 ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc tccgggggat
      601 cttggtggcg tgaaactccc gcacctcttc ggccagcgcc ttgtagaagc gcgtatggct
      661 tctagctatc cttatgacgt gcctgactat gccagcctgg gaggaccttc tagcggatcc
      721 accatgactg agtacaaact ggtggtggtt ggagcaggtg gtgttgggaa aagcgcactg
      781 acaatccagc taatccagaa ccactttgta gatgaatatg atcccaccat agaggattct
      841 tacagaaaac aagtggttat agatggtgaa acctgtttgt tggacatact ggatacagct
      901 ggacaagaag agtacagtgc catgagagac caatacatga ggacaggcga aggcttcctc
      961 tgtgtatttg ccatcaataa tagcaagtca tttgcggata ttaacctcta cagggagcag
     1021 attaagcgag taaaagactc ggatgatgta cctatggtgc tagtgggaaa caagtgtgat
     1081 ttgccaacaa ggacagttga tacaaaacaa gcccacgaac tggccaagag ttacgggatt
     1141 ccattcattg aaacctcagc caagaccaga cagggtgttg aagatgcttt ttacacactg
     1201 gtaagagaaa tacgccagta ccgaatgaaa aaactcaaca gcagtgatga tgggactcag
     1261 ggttgtatgg gattgccatg tgtggtgatg taagtcgacg aattcggatc ctgagaactt
     1321 cagggtgagt ttggggaccc ttgattgttc tttctttttc gctattgtaa aattcatgtt
     1381 atatggaggg ggcaaagttt tcagggtgtt gtttagaatg ggaagatgtc ccttgtatca
     1441 ccatggaccc tcatgataat tttgtttctt tcactttcta ctctgttgac aaccattgtc
     1501 tcctcttatt ttcttttcat tttctgtaac tttttcgtta aactttagct tgcatttgta
     1561 acgaattttt aaattcactt ttgtttattt gtcagattgt aagtactttc tctaatcact
     1621 tttttttcaa ggcaatcagg gtatattata ttgtacttca gcacagtttt agagaacaat
     1681 tgttataatt aaatgataag gtagaatatt tctgcatata aattctggct ggcgtggaaa
     1741 tattcttatt ggtagaaaca actacaccct ggtcatcatc ctgcctttct ctttatggtt
     1801 acaatgatat acactgtttg agatgaggat aaaatactct gagtccaaac cgggcccctc
     1861 tgctaaccat gttcatgcct tcttctcttt cctacagctc ctgggcaacg tgctggttgt
     1921 tgtgctgtct catcattttg gcaaagaatt cactcctcag gtgcaggctg cctatcagaa
     1981 ggtggtggct ggtgtggcca atgccctggc tcacaaatac cactgagatc tttttccctc
     2041 tgccaaaaat tatggggaca tcatgaagcc ccttgagcat ctgacttctg gctaataaag
     2101 gaaatttatt ttcattgcaa tagtgtgttg gaattttttg tgtctctcac tcggaaggac
     2161 atatgggagg gcaaatcatt taaaacatca gaatgagtat ttggtttaga gtttggcaac
     2221 atatgcccat atgctggctg ccatgaacaa aggttggcta taaagaggtc atcagtatat
     2281 gaaacagccc cctgctgtcc attccttatt ccatagaaaa gccttgactt gaggttagat
     2341 tttttttata ttttgttttg tgttattttt ttctttaaca tccctaaaat tttccttaca
     2401 tgttttacta gccagatttt tcctcctctc ctgactactc ccagtcatag ctgtccctct
     2461 tctcttatgg agatccctcg acggatcggg agatctcccg atccccggga attctgtgga
     2521 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     2581 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     2641 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     2701 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatgca tggctgacta
     2761 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
     2821 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc ttggccacca tgggaaagcc
     2881 tgagctgacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga
     2941 cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg
     3001 tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta
     3061 tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag
     3121 cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc
     3181 tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc
     3241 ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata
     3301 cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac
     3361 tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg
     3421 ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt
     3481 cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga
     3541 ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca
     3601 gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc
     3661 gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga
     3721 tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt
     3781 cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt
     3841 actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg aatagagtag
     3901 atgccgaccg aacaagagct gatttcgaga acgcctcagc cagcaactcg cgcgagccta
     3961 gcaaggcaaa tgcgagagaa cggccttacg cttggtggca cagttctcgt ccacagttcg
     4021 ctaagctcgc tcggctgggt cgcgggaggg ccggtcgcag tgattcaggc ccttctggat
     4081 tgtgttggtc cccagggcac gattgtcatg cccacgcact cgggtgatct gactgatccc
     4141 gcagattgga gatcgccgcc cgtgcctgcc gattgggtgc agatcagcct cgaggcgatt
     4201 agtccaattt gttaaagaca ggatatcagt ggtccaggct ctagttttga ctcaacaata
     4261 tcaccagctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca
     4321 gggccaagaa cagatggtcc ccagatgcgg tccagccctc agcagtttct agagaaccat
     4381 cagatgtttc cagggtgccc caaggacctg aaatgaccct gtgccttatt tgaactaacc
     4441 aatcagttcg cttctcgctt ctgttcgcgc gcttctgctc cccgagctca ataaaagagc
     4501 ccacaacccc tcactcgggg cgccagtcct ccgattgact gagtcgcccg ggtacccgtg
     4561 tatccaataa accctcttgc agttgcatcc gacttgtggt ctcgctgttc cttgggaggg
     4621 tctcctctga gtgattgact acccgtcagc gggggtcttt cactgggaga atccactgaa
     4681 caaccaaaaa gtgacctgca tcttagtgta actcctgctt tcccatatag ctccagagga
     4741 gggcccattt cccatcagct tggcttcctg gattccggtg tttcagagct cccagtgacc
     4801 caggctgtag ccctgccaat gccaccttaa aacaaaacac tccttcttta cctttactcc
     4861 ctagaaacat tgctttccca tcccagaaca gcagtcgtga agaaaacaga tcatcagtgt
     4921 cctgcactca gactcttgcg gtcgaggagc tttttgcaaa agcctaggcc tccaaaaaag
     4981 cctcttcact acttctggaa tagctcagag gccgaggcgg cctcggcctc tgcataaata
     5041 aaaaaaatta gtcagccatg gggcggagaa tgggcggaac tgggcggagt taggggcggg
     5101 atgggcggag ttaggggcgg gactatggtt gctgactaat tgctgcatta atgaatcggc
     5161 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     5221 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     5281 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     5341 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     5401 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     5461 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     5521 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     5581 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     5641 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     5701 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     5761 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     5821 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5881 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     5941 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     6001 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     6061 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     6121 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     6181 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     6241 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     6301 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     6361 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     6421 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     6481 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     6541 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     6601 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     6661 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     6721 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     6781 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     6841 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     6901 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     6961 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     7021 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     7081 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa
     7141 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc
     7201 gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca
     7261 gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt
     7321 ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac
     7381 catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggaaattg
     7441 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     7501 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     7561 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     7621 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     7681 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     7741 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     7801 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     7861 ccgcgcttaa tgcgccgcta cagggcgcgt ccattcgcca ttcaggctgc gcaactgttg
     7921 ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc
     7981 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac
     8041 ggccagtgaa ttgtaatacg actcactata gggcgaattc gagctcgccc
//
