LOCUS       sequence-9358-ex        7619 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 7619)
  AUTHORS   Stern P, Astrof S, Erkeland SJ, Schustak J, Sharp PA, Hynes RO
  TITLE     A system for Cre-regulated RNA interference in vivo.
  JOURNAL   Proc Natl Acad Sci U S A. 2008 Sep 16. 105(37):13895-900.
  PUBMED    18779577
REFERENCE   2  (bases 1 to 7619)
  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: "Proc Natl 
            Acad Sci U S A. 2008 Sep 16. 105(37):13895-900."
COMMENT     Sequence Label: MSCV P2Gm p53
FEATURES             Location/Qualifiers
     source          1..7619
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    580..921
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             988..1404
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1334..1356
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1372..1388
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     regulatory      1430..1439
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1436..2032
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MVEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     1932..1952
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             2054..2119
                     /codon_start=1
                     /product="2A peptide from foot-and-mouth disease virus 
                     polyprotein"
                     /label=F2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="VKQTLNFDLLKLAGDVESNPGP"
     regulatory      2129..2138
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2135..2137,2138..2140,2141..2854)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2135..2137,2138..2140,2141..2851)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2180..2201)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2441..2460)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2788..2809
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     ncRNA           2904..3025
                     /label=5' miR-30a
                     /note="sequence upstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           3053..3067
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           3095..3221
                     /label=3' miR-30a
                     /note="sequence downstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     misc_feature    3287..3875
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3340..3360)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3758..3769)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3932..4447
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     primer_bind     complement(4616..4632)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4616..4632)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4629..4651)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4640..4656
                     /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(4664..4670,4671..4688,4689..4694))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4709..4730
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4847..4864)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5018..5606)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5098..5117)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5777..6568,6569..6637))
                     /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     6400..6419
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6638..6742)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6810..6828
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6866..6888)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6988..7007
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7201..7223
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7399..7418
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(7618..7619,1..516)
                     /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 gaaaatgacc ctgtgcctta tttgaactaa ccaatcagtt
      301 cgcttctcgc ttctgttcgc gcgcttctgc tccccgagct caataaaaga gcccacaacc
      361 cctcactcgg cgcgccagtc ctccgataga ctgcgtcgcc cgggtacccg tattcccaat
      421 aaagcctctt gctgtttgca tccgaatcgt ggactcgctg atccttggga gggtctcctc
      481 agattgattg actgcccacc tcgggggtct ttcatttgga ggttccaccg agatttggag
      541 acccctgcct agggaccacc gacccccccg ccgggaggta agctggccag cggtcgtttc
      601 gtgtctgtct ctgtctttgt gcgtgtttgt gccggcatct aatgtttgcg cctgcgtctg
      661 tactagttag ctaactagct ctgtatctgg cggacccgtg gtggaactga cgagttctga
      721 acacccggcc gcaaccctgg gagacgtccc agggactttg ggggccgttt ttgtggcccg
      781 acctgaggaa gggagtcgat gtggaatccg accccgtcag gatatgtggt tctggtagga
      841 gacgagaacc taaaacagtt cccgcctccg tctgaatttt tgctttcggt ttggaaccga
      901 agccgcgcgt cttgtctgct gcagcgctgc agcatcgttc tgtgttgtct ctgtctgact
      961 gtgtttctgt atttgtctga aaattagggc cagactgtta ccactccctt aagtttgacc
     1021 ttaggtcact ggaaagatgt cgagcggatc gctcacaacc agtcggtaga tgtcaagaag
     1081 agacgttggg ttaccttctg ctctgcagaa tggccaacct ttaacgtcgg atggccgcga
     1141 gacggcacct ttaaccgaga cctcatcacc caggttaaga tcaaggtctt ttcacctggc
     1201 ccgcatggac acccagacca ggtcccctac atcgtgacct gggaagcctt ggcttttgac
     1261 ccccctccct gggtcaagcc ctttgtacac cctaagcctc cgcctcctct tcctccatcc
     1321 gccccgtctc tcccccttga acctcctcgt tcgaccccgc ctcgatcctc cctttatcca
     1381 gccctcactc cttctctagg cgccggaatt agatctctcg actaccggtg ccaccatggt
     1441 ggagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac
     1501 cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca
     1561 catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg
     1621 caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt
     1681 cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg
     1741 gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc
     1801 gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc
     1861 cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac
     1921 ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt
     1981 cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg ccaaacagaa
     2041 aattgtggca ccagtgaaac agactttgaa ttttgacctt ctcaagttgg cgggagacgt
     2101 cgagtccaac cctgggcccg gcccggtcgc caccatggtg agcaagggcg aggagctgtt
     2161 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     2221 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     2281 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
     2341 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     2401 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     2461 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     2521 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     2581 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
     2641 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     2701 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag
     2761 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     2821 gatcactctc ggcatggacg agctgtacaa gtagcggccg caagccttgt taagtgctcg
     2881 cttcggcagc acatatacta tgtttgaatg aggcttcagt actttacaga atcgttgcct
     2941 gcacatcttg gaaacacttg ctgggattac ttcttcaggt taacccaaca gaaggctcga
     3001 gaaggtatat tgctgttgac agtgagcgcc cactacaagt acatgtgtaa tagtgaagcc
     3061 acagatgtat tacacatgta cttgtagtgg atgcctactg cctcggaatt caaggggcta
     3121 ctttaggagc aattatcttg tttactaaaa ctgaatacct tgctatctct ttgatacatt
     3181 tttacaaagc tgaattaaaa tggtataaat taaatcactt ttttcaattg gaagactaat
     3241 gcgtttaaac ctcgacctgc agccaagctt atcgataatt cccgataatc aacctctgga
     3301 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
     3361 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
     3421 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
     3481 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
     3541 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
     3601 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     3661 ttccgtggtg ttgtcgggga agctgacgtc ctttccatgg ctgctcgcct gtgttgccac
     3721 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
     3781 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca
     3841 gacgagtcgg atctcccttt gggccgcctc cccgcatcgg gaattatcga taaaataaaa
     3901 gattttattt agtctccaga aaaagggggg aatgaaagac cccacctgta ggtttggcaa
     3961 gctagcttaa gtaacgccat tttgcaaggc atggaaaata cataactgag aatagagaag
     4021 ttcagatcaa ggttaggaac agagagacag cagaatatgg gccaaacagg atatctgtgg
     4081 taagcagttc ctgccccggc tcagggccaa gaacagatgg tccccagatg cggtcccgcc
     4141 ctcagcagtt tctagagaac catcagatgt ttccagggtg ccccaaggac ctgaaaatga
     4201 ccctgtgcct tatttgaact aaccaatcag ttcgcttctc gcttctgttc gcgcgcttct
     4261 gctccccgag ctcaataaaa gagcccacaa cccctcactc ggcgcgccag tcctccgata
     4321 gactgcgtcg cccgggtacc cgtgtatcca ataaaccctc ttgcagttgc atccgacttg
     4381 tggtctcgct gttccttggg agggtctcct ctgagtgatt gactacccgt cagcgggggt
     4441 ctttcatggg taacagtttc ttgaagttgg agaacaacat tctgagggta ggagtcgaat
     4501 attaagtaat cctgactcaa ttagccactg ttttgaatcc acatactcca atactcctga
     4561 aatagttcat tatggacagc gcagaagagc tggggagaat taattcgtaa tcatggtcat
     4621 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     4681 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     4741 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     4801 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     4861 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     4921 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     4981 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     5041 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     5101 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     5161 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     5221 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     5281 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     5341 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     5401 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     5461 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     5521 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     5581 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     5641 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     5701 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     5761 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     5821 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     5881 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     5941 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     6001 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     6061 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     6121 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     6181 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     6241 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6301 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     6361 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     6421 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     6481 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     6541 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     6601 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     6661 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     6721 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     6781 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg
     6841 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     6901 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     6961 gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc
     7021 atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgccatt
     7081 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
     7141 gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt
     7201 cccagtcacg acgttgtaaa acgacggcgc aaggaagcag cccagtagta ggttgaggcc
     7261 gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc
     7321 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg
     7381 agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc
     7441 gccggtgatg ccggccacga tgcgtccggc gtagaggcga ttagtccaat ttgttaaaga
     7501 caggatatca gtggtccagg ctctagtttt gactcaacaa tatcaccagc tgaagcctat
     7561 agagtacgag ccatagataa aataaaagat tttatttagt ctccagaaaa aggggggaa
//
