LOCUS       sequence-9357-ex        7618 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 7618)
  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 7618)
  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 FF
FEATURES             Location/Qualifiers
     source          1..7618
                     /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           3052..3066
                     /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           3094..3220
                     /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    3286..3874
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3339..3359)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3757..3768)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3931..4446
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     primer_bind     complement(4615..4631)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4615..4631)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4628..4650)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4639..4655
                     /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(4663..4669,4670..4687,4688..4693))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4708..4729
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5017..5605)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5097..5116)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5776..6567,6568..6636))
                     /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     6399..6418
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6637..6741)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6809..6827
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6865..6887)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6987..7006
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7200..7222
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7398..7417
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(7617..7618,1..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 agtgagcgag ctcccgtgaa ttggaatcct agtgaagcca
     3061 cagatgtagg attccaattc agcgggagcc tgcctactgc ctcggaattc aaggggctac
     3121 tttaggagca attatcttgt ttactaaaac tgaatacctt gctatctctt tgatacattt
     3181 ttacaaagct gaattaaaat ggtataaatt aaatcacttt tttcaattgg aagactaatg
     3241 cgtttaaacc tcgacctgca gccaagctta tcgataattc ccgataatca acctctggat
     3301 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
     3361 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
     3421 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg
     3481 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc
     3541 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa
     3601 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
     3661 tccgtggtgt tgtcggggaa gctgacgtcc tttccatggc tgctcgcctg tgttgccacc
     3721 tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt
     3781 ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag
     3841 acgagtcgga tctccctttg ggccgcctcc ccgcatcggg aattatcgat aaaataaaag
     3901 attttattta gtctccagaa aaagggggga atgaaagacc ccacctgtag gtttggcaag
     3961 ctagcttaag taacgccatt ttgcaaggca tggaaaatac ataactgaga atagagaagt
     4021 tcagatcaag gttaggaaca gagagacagc agaatatggg ccaaacagga tatctgtggt
     4081 aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc ggtcccgccc
     4141 tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc tgaaaatgac
     4201 cctgtgcctt atttgaacta accaatcagt tcgcttctcg cttctgttcg cgcgcttctg
     4261 ctccccgagc tcaataaaag agcccacaac ccctcactcg gcgcgccagt cctccgatag
     4321 actgcgtcgc ccgggtaccc gtgtatccaa taaaccctct tgcagttgca tccgacttgt
     4381 ggtctcgctg ttccttggga gggtctcctc tgagtgattg actacccgtc agcgggggtc
     4441 tttcatgggt aacagtttct tgaagttgga gaacaacatt ctgagggtag gagtcgaata
     4501 ttaagtaatc ctgactcaat tagccactgt tttgaatcca catactccaa tactcctgaa
     4561 atagttcatt atggacagcg cagaagagct ggggagaatt aattcgtaat catggtcata
     4621 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     4681 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     4741 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     4801 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     4861 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     4921 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     4981 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     5041 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     5101 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     5161 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     5221 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     5281 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     5341 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     5401 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     5461 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     5521 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     5581 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     5641 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     5701 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     5761 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     5821 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     5881 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     5941 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     6001 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     6061 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     6121 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     6181 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     6241 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     6301 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     6361 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     6421 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     6481 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     6541 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     6601 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     6661 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     6721 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac
     6781 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc
     6841 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     6901 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     6961 cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca
     7021 tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgccattc
     7081 gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg
     7141 ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc
     7201 ccagtcacga cgttgtaaaa cgacggcgca aggaagcagc ccagtagtag gttgaggccg
     7261 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     7321 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     7381 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     7441 ccggtgatgc cggccacgat gcgtccggcg tagaggcgat tagtccaatt tgttaaagac
     7501 aggatatcag tggtccaggc tctagttttg actcaacaat atcaccagct gaagcctata
     7561 gagtacgagc catagataaa ataaaagatt ttatttagtc tccagaaaaa ggggggaa
//
