LOCUS       sequence-119955-        7648 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The prostate specific promoter controls expression of FlpO linked to
            a blue flourescent protein. gRNA towards PTEN ex5, p53 ex8 and SMAD4
            ex2. are expressed by the U6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7648)
  TITLE     Cas9/CRISPR pig transposon 
            plasmid_LIR_TRE_CAG_mKateII-Puro-STOP_mVenus-Cas9-PA-RIR
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7648)
  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: 
            "Unpublished"
COMMENT     Sequence Label: 3xgRNA: PTEN exon 5, p53 exon 8, SMAD4 exon 2
            ...
FEATURES             Location/Qualifiers
     source          1..7648
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        698..946
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        967..1042
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1055..1303
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1324..1399
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1412..1660
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1681..1756
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     regulatory      2256..2265
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2262..2264,2265..2267,2268..2978)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Goedhart et 
                     al., 2012)"
                     /label=mTurquoise2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2307..2328)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2568..2587)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2915..2936
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2994..3050
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(3057..3062,3063..3083,3084..4355)
                     /codon_start=1
                     /product="nuclear-targeted site-specific recombinase"
                     /label=FLPo
                     /note="FLPo is a mouse codon-optimized fusion between the 
                     SV40 nuclear localization signal and a thermostable version
                     of the Saccharomyces cerevisiae site-specific recombinase 
                     FLP.  Recombination occurs at FRT sequences."
                     /translation="MAPKKKRKVMSQFDILCKTPPKVLVRQFVERFERPSGEKIASCAA
                     ELTYLCWMITHNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKK
                     LIPAWEFTIIPYNGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESI
                     WEITEKILNSFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGV
                     IIQCLVTETKTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEY
                     QLLKDNLVRSYNKALKKNAPYPIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWS
                     DKRASAVARTTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQL
                     KGSAEGSIRYPAWNGIISQEVLDYLSSYINRRI"
     polyA_signal    4459..4580
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4496..4515)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4664..4681)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4670..4894
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(4911..4944)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     primer_bind     complement(5427..5443)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5427..5443)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5440..5462)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5451..5467
                     /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(5475..5481,5482..5499,5500..5505))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5520..5541
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5658..5675)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5829..6417)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5909..5928)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6588..7379,7380..7448))
                     /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     7211..7230
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7449..7553)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7621..7639
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cagatctgat atctctagag
      421 aagtcgacgt ttaaagtacc ctacagttga agtcggaagt ttacatacac ttaagttgga
      481 gtcattaaaa ctcgtttttc aactactcca caaatttctt gttaacaaac aatagttttg
      541 gcaagtcagt taggacatct actttgtgca tgacacaagt catttttcca acaattgttt
      601 acagacagat tatttcactt ataattcact gtatcacaat tccagtgggt cagaagttta
      661 catacactaa gttgactgtg cctttaaaca gtcgcgagag ggcctatttc ccatgattcc
      721 ttcatatttg catatacgat acaaggctgt tagagagata attggaatta atttgactgt
      781 aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt
      841 tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta
      901 tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgcag caattcactg
      961 taaagcgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
     1021 aaaagtggca ccgagtcggt gcttttttac cggtgagggc ctatttccca tgattccttc
     1081 atatttgcat atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa
     1141 cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc
     1201 agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt
     1261 cgatttcttg gctttatata tcttgtggaa aggacgaaac accgtgcgtg tttgtgcctg
     1321 tccgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
     1381 agtggcaccg agtcggtgct tttttatcga tgagggccta tttcccatga ttccttcata
     1441 tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac
     1501 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
     1561 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
     1621 tttcttggct ttatatatct tgtggaaagg acgaaacacc gaaggagaaa aaagatgaat
     1681 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
     1741 ggcaccgagt cggtgctttt ttggtaccac tagttatgat agcatcttgt tcttagtctt
     1801 tttcttaata gggacataaa gcccacaaat aaaaatatgc ctgaagaatg ggacaggcat
     1861 tgggcattgt ccatgcctag taaagtactc caagaaccta tttgtatact agatgacaca
     1921 atgtaatagt catcatgttt aaacatctac cattccagtt aagaaaatat gatagcatct
     1981 tgttcttagt ctttttctta atagggacat aaagcccaca aataaaaata tgcctgaaga
     2041 atgggacagg cattgggcat tgtccatgcc tagtaaagta ctccaagaac ctatttgtat
     2101 actagatgac acaatgtcaa tgtctgtgta caactgccaa ctgggatgca agacactgcc
     2161 catgccaatc atcctgaaaa gcagctataa aaagcaggaa gctactctgc accttgtcag
     2221 tgaggtccag atacctacag attaattaac ccggggccac catggtgagc aagggcgagg
     2281 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     2341 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     2401 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgtcct
     2461 ggggcgtgca gtgcttcgcc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     2521 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     2581 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     2641 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactact
     2701 ttagcgacaa cgtctatatc accgccgaca agcagaagaa cggcatcaag gccaacttca
     2761 agatccgcca caacatcgag gacggcggcg tgcagctcgc cgaccactac cagcagaaca
     2821 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtcca
     2881 agctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     2941 ccgccgggat cactctcggc atggacgagc tgtacaaggt atacggaagc ggagctacta
     3001 acttcagcct gctgaagcag gctggagacg tggaggagaa ccctggacct gctagcatgg
     3061 ctcctaagaa gaagaggaag gtgatgagcc agttcgacat cctgtgcaag acccccccca
     3121 aggtgctggt gcggcagttc gtggagagat tcgagaggcc cagcggcgag aagatcgcca
     3181 gctgtgccgc cgagctgacc tacctgtgct ggatgatcac ccacaacggc accgccatca
     3241 agagggccac cttcatgagc tacaacacca tcatcagcaa cagcctgagc ttcgacatcg
     3301 tgaacaagag cctgcagttc aagtacaaga cccagaaggc caccatcctg gaggccagcc
     3361 tgaagaagct gatccccgcc tgggagttca ccatcatccc ttacaacggc cagaagcacc
     3421 agagcgacat caccgacatc gtgtccagcc tgcagctgca gttcgagagc agcgaggagg
     3481 ccgacaaggg caacagccac agcaagaaga tgctgaaggc cctgctgtcc gagggcgaga
     3541 gcatctggga gatcaccgag aagatcctga acagcttcga gtacaccagc aggttcacca
     3601 agaccaagac cctgtaccag ttcctgttcc tggccacatt catcaactgc ggcaggttca
     3661 gcgacatcaa gaacgtggac cccaagagct tcaagctggt gcagaacaag tacctgggcg
     3721 tgatcattca gtgcctggtg accgagacca agacaagcgt gtccaggcac atctactttt
     3781 tcagcgccag aggcaggatc gaccccctgg tgtacctgga cgagttcctg aggaacagcg
     3841 agcccgtgct gaagagagtg aacaggaccg gcaacagcag cagcaacaag caggagtacc
     3901 agctgctgaa ggacaacctg gtgcgcagct acaacaaggc cctgaagaag aacgccccct
     3961 accccatctt cgctatcaag aacggcccta agagccacat cggcaggcac ctgatgacca
     4021 gctttctgag catgaagggc ctgaccgagc tgacaaacgt ggtgggcaac tggagcgaca
     4081 agagggcctc cgccgtggcc aggaccacct acacccacca gatcaccgcc atccccgacc
     4141 actacttcgc cctggtgtcc aggtactacg cctacgaccc catcagcaag gagatgatcg
     4201 ccctgaagga cgagaccaac cccatcgagg agtggcagca catcgagcag ctgaagggca
     4261 gcgccgaggg cagcatcaga taccccgcct ggaacggcat catcagccag gaggtgctgg
     4321 actacctgag cagctacatc aacaggcgga tctaaatgca tcagccatac cacatttgta
     4381 gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg
     4441 aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     4501 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     4561 aaactcatca atgtatctta acgcgtaaat tgtaagcggg ccggccatat ttgtcaccgg
     4621 actgcggccg cgactctaga tcataatcag cccgggggat cagcctcgac tgtgccttct
     4681 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     4741 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     4801 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     4861 agcaggcatg ctggggatgc ggtgggctct atggaaccag ctggggcgcg ataacttcgt
     4921 ataggagact ttatacgaag ttatgcgcca ttctagttgt ggtttgtcca aactcatcaa
     4981 tgtatcttat catgtctgga tcccatcaca aagctctgac ctcaatccta tagaaaggag
     5041 gaatgagcca aaattcaccc aacttattgt gggaagcttg tggaaggcta ctcgaaatgt
     5101 ttgacccaag ttaaacaatt taaaggcaat gctaccaaat actaattgag tgtatgttaa
     5161 cttctgaccc actgggaatg tgatgaaaga aataaaagct gaaatgaatc attctctcta
     5221 ctattattct gatatttcac attcttaaaa taaagtggtg atcctaactg accttaagac
     5281 agggaatctt tactcggatt aaatgtcagg aattgtgaaa aagtgagttt aaatgtattt
     5341 ggctaaggtg tatgtaaact tccgacttca actgtagggg atcctctaga gtcgacctgc
     5401 aggcatgcaa gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     5461 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     5521 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     5581 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     5641 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     5701 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     5761 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     5821 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     5881 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     5941 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     6001 tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc
     6061 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     6121 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     6181 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     6241 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     6301 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     6361 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     6421 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     6481 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     6541 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     6601 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     6661 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     6721 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     6781 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     6841 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     6901 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     6961 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     7021 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     7081 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     7141 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     7201 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     7261 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     7321 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     7381 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     7441 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     7501 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     7561 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa
     7621 aataggcgta tcacgaggcc ctttcgtc
//
