LOCUS       sequence-257660-        6526 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 6526)
  AUTHORS   Ikeda M, Kawata A, Nishikawa M, Tateishi Y, Yamaguchi M, Nakagawa K,
            Hirabayashi S, Bao Y, Hidaka S, Hirata Y, Hata Y
  TITLE     Hippo pathway-dependent and -independent roles of RASSF6.
  JOURNAL   Sci Signal. 2009 Sep 29;2(90):ra59.
  PUBMED    19797269
REFERENCE   2  (bases 1 to 6526)
  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: "Sci 
            Signal."; date: "2009-09-29"; volume: "2(90)"; pages: "ra59"
COMMENT     Sequence Label: pClneoFH human RASSF1A
FEATURES             Location/Qualifiers
     source          1..6526
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        37..416
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        417..628
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     579..599
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     625..649
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          789..921
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     904..923
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        966..984
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1000..1023
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1030..1047
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    2129..2250
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2140..2159)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2194..2213
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      2436..2891
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2523..2542)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2733..2754
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3008..3365
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3008..3199
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3031..3051)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3216..3351
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3416..4210
                     /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(3470..3489)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4080..4099
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4274..4322
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(4363..4382)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4482..4504
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4542..4560)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4628..4732
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4733..4801,4802..5593)
                     /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(4951..4970)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5764..6352
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6253..6272
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tttatattgg ctcatgtcca atatgaccgc catgttggca ttgattattg actagttatt
       61 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
      121 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
      181 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
      241 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtccgc
      301 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
      361 tacgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga
      421 tgcggttttg gcagtacacc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa
      481 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc
      541 caaaatgtcg taacaactgc gatcgcccgc cccgttgacg caaatgggcg gtaggcgtgt
      601 acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatca ctagaagctt
      661 tattgcggta gtttatcaca gttaaattgc taacgcagtc agtgcttctg acacaacagt
      721 ctcgaactta agctgcagtg actctcttaa ggtagccttg cagaagttgg tcgtgaggca
      781 ctgggcaggt aagtatcaag gttacaagac aggtttaagg agaccaatag aaactgggct
      841 tgtcgagaca gagaagactc ttgcgtttct gataggcacc tattggtctt actgacatcc
      901 actttgcctt tctctccaca ggtgtccact cccagttcaa ttacagctct taaggctaga
      961 gtacttaata cgactcacta taggctagcc ggggccatgg actacaaaga cgatgacgac
     1021 aaggaattgc accatcatca tcatcatgaa ttctcggggg agcctgagct cattgagctg
     1081 cgggagctgg cacccgctgg gcgcgctggg aagggccgca cccggctgga gcgtgccaac
     1141 gcgctgcgca tcgcgcgggg caccgcgtgc aaccccacac ggcagctggt ccctggccgt
     1201 ggccaccgct tccagcccgc ggggcccgcc acgcacacgt ggtgcgacct ctgtggcgac
     1261 ttcatctggg gcgtcgtgcg caaaggcctg cagtgcgcgc attgcaagtt cacctgccac
     1321 taccgctgcc gcgcgctcgt ctgcctggac tgttgcgggc cccgggacct gggctgggaa
     1381 cccgcggtgg agcgggacac gaacgtggac gagcctgtgg agtgggagac acctgacctt
     1441 tctcaagctg agattgagca gaagatcaag gagtacaatg cccagatcaa cagcaacctc
     1501 ttcatgagct tgaacaagga cggttcttac acaggcttca tcaaggttca gctgaagctg
     1561 gtgcgccctg tctctgtgcc ctccagcaag aagccaccct ccttgcagga tgcccggcgg
     1621 ggcccaggac ggggcacaag tgtcaggcgc cgcacttcct tttacctgcc caaggatgct
     1681 gtcaagcacc tgcatgtgct gtcacgcaca agggcacgtg aagtcattga ggccctgctg
     1741 cgaaagttct tggtggtgga tgacccccgc aagtttgcac tctttgagcg cgctgagcgt
     1801 cacggccaag tgtacttgcg gaagctgttg gatgatgagc agcccctgcg gctgcggctc
     1861 ctggcagggc ccagtgacaa ggccctgagc tttgtcctga aggaaaatga ctctggggag
     1921 gtgaactggg acgccttcag catgcctgaa ctacataact tcctacgtat cctgcagcgg
     1981 gaggaggagg agcacctccg ccagatcctg cagaagtact cctattgccg ccagaagatc
     2041 caagaggccc tgcacgcctg cccccttggg tagtcgaccc gggcggccgc ttccctttag
     2101 tgagggttaa tgcttcgagc agacatgata agatacattg atgagtttgg acaaaccaca
     2161 actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt
     2221 gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt
     2281 caggttcagg gggagatgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt
     2341 aaaatccgat aaggatcgat ccgggctggc gtaatagcga agaggcccgc accgatcgcc
     2401 cttcccaaca gttgcgcagc ctgaatggcg aatggacgcg ccctgtagcg gcgcattaag
     2461 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     2521 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     2581 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     2641 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     2701 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     2761 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     2821 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     2881 gcttacaatt tcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     2941 atacgcggat ctgcgcagca ccatggcctg aaataacctc tgaaagagga acttggttag
     3001 gtaccttctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt
     3061 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3121 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3181 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3241 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3301 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     3361 gcaaaaagct tgattcttct gacacaacag tctcgaactt aaggctagag ccaccatgat
     3421 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     3481 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     3541 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga
     3601 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     3661 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     3721 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     3781 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     3841 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     3901 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga
     3961 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     4021 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     4081 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     4141 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     4201 gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca
     4261 tcacgatggc cgcaataaaa tatctttatt ttcattacat ctgtgtgttg gttttttgtg
     4321 tgaatcgata gcgataagga tccgcgtatg gtgcactctc agtacaatct gctctgatgc
     4381 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     4441 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     4501 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
     4561 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     4621 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     4681 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     4741 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     4801 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     4861 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     4921 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     4981 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     5041 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     5101 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     5161 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     5221 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     5281 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     5341 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     5401 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     5461 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     5521 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     5581 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     5641 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     5701 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     5761 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     5821 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     5881 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     5941 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     6001 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     6061 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     6121 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     6181 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     6241 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     6301 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     6361 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tggctcgaca
     6421 gatcttcaat attggccatt agccatatta ttcattggtt atatagcata aatcaatatt
     6481 ggctattggc cattgcatac gttgtatcta tatcataata tgtaca
//
