LOCUS       sequence-325441-        6210 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Introduce Dox-inducible (TRE promoter) GFP cDNA plus miR30-embedded 
            shPten into (ES) cells by recombination-mediated cassette exchange.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6210)
  TITLE     Karreth lab plasmids
REFERENCE   2  (bases 1 to 6210)
  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"
COMMENT     Sequence Label: cTRE-GFP-miR30_shPten.1523
FEATURES             Location/Qualifiers
     source          1..6210
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    join(25..90,91..128)
                     /label=Ad SA
                     /note="adenovirus major late transcript splice acceptor 
                     (Friedrich and Soriano, 1991)"
     polyA_signal    297..431
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    551..685
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    join(697..715,716..738,739..757,758..780,781..799,800..822,
                     823..841,842..864,865..883,884..906,907..925,926..948,
                     949..967)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     promoter        1000..1038
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1035..1059
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(1144..1146,1147..1149,1150..1863)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANLKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1189..1210)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1450..1469)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1797..1818
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     ncRNA           2057..2071
                     /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"
     intron          2239..2811
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     rep_origin      complement(3139..3727)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3219..3238)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3898..4689,4690..4758))
                     /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     4521..4540
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4759..4863)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4889..5344)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5026..5047)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5238..5257
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5471..5493
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5485..5502
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5486..5502
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5509..5528
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5509..5527
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     5560..5576
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5560..5576
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        5620..6119
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(5655..5678)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     6052..6071
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     protein_bind    join(6165..6210,1..2)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
ORIGIN
        1 tcgatccccc gggctgcaga tctgtagggc gcagtagtcc agggtttcct tgatgatgtc
       61 atacttatcc tgtccctttt ttttccacag ctcgcggttg aggacaaact cttcgcggtc
      121 tttccagtgg ggatcgacgg tatcgataag cttgcagatc tgcgactcta gaggatctgc
      181 gactctagag gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa
      241 acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact
      301 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
      361 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
      421 atgtctggat ctgcgactct agaggatcat aatcagccat accacatttg tagaggtttt
      481 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
      541 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
      601 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
      661 caatgtatct tatcatgtct ggatcgagtt taccactccc tatcagtgat agagaaaagt
      721 gaaagtcgag tttaccactc cctatcagtg atagagaaaa gtgaaagtcg agtttaccac
      781 tccctatcag tgatagagaa aagtgaaagt cgagtttacc actccctatc agtgatagag
      841 aaaagtgaaa gtcgagttta ccactcccta tcagtgatag agaaaagtga aagtcgagtt
      901 taccactccc tatcagtgat agagaaaagt gaaagtcgag tttaccactc cctatcagtg
      961 atagagaaaa gtgaaagtcg agctcggtac ccgggtcgag gtaggcgtgt acggtgggag
     1021 gcctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc
     1081 tgttttgacc tccatagaag acaccgggac cgatccagcc tccgcggccc cgaattgacc
     1141 accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     1201 gacggcgacg taaacggcca caagttcagc gtgtctggcg agggcgaggg cgatgccacc
     1261 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     1321 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
     1381 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     1441 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     1501 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     1561 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     1621 aacggcatca aggcgaacct caagatccgc cacaacatcg aggacggcag cgtgcagctc
     1681 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     1741 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     1801 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     1861 taatgacgta cggcggccgc ctcgactagg gataacaggg taattgtttg aatgaggctt
     1921 cagtacttta cagaatcgtt gcctgcacat cttggaaaca cttgctggga ttacttcgac
     1981 ttcttaaccc aacagaaggc tcgagaaggt atattgctgt tgacagtgag cgaccagcta
     2041 aaggtgaaga tatatagtga agccacagat gtatatatct tcacctttag ctggctgcct
     2101 actgcctcgg aattcgagca attatcttgt ttactaaaac tgaatacctt gctatctctt
     2161 tgatacattt ttacaaagct gaattaaaat ggtataaatt aaatcacttt tttcaattga
     2221 cgcgttgaga acttcagggt gagtttgggg acccttgatt gttctttctt tttcgctatt
     2281 gtaaaattca tgttatatgg agggggcaaa gttttcaggg tgttgtttag aatgggaaga
     2341 tgtcccttgt atcaccatgg accctcatga taattttgtt tctttcactt tctactctgt
     2401 tgacaaccat tgtctcctct tattttcttt tcattttctg taactttttc gttaaacttt
     2461 agcttgcatt tgtaacgaat ttttaaattc acttttgttt atttgtcaga ttgtaagtac
     2521 tttctctaat cacttttttt tcaaggcaat cagggtatat tatattgtac ttcagcacag
     2581 ttttagagaa caattgttat aattaaatga taaggtagaa tatttctgca tataaattct
     2641 ggctggcgtg gaaatattct tattggtaga aacaactaca ccctggtcat catcctgcct
     2701 ttctctttat ggttacaatg atatacactg tttgagatga ggataaaata ctctgagtcc
     2761 aaaccgggcc cctctgctaa ccatgttcat gccttcttct ctttcctaca gctcctgggc
     2821 aacgtgctgg ttgttgtgct gtctcatcat tttggcaaag gattcactcc tcaggtgcag
     2881 gctgcctatc agaaggtggt ggctggtgtg gccaatgccc tggctcacaa ataccactga
     2941 gatcgttttc cctctgccaa aaattatggg gacatcatga agccccttga gcatctgact
     3001 tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt ctcgatcgct
     3061 agcatcctag gcgcgccaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     3121 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     3181 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     3241 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     3301 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     3361 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     3421 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     3481 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     3541 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     3601 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     3661 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     3721 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     3781 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     3841 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     3901 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     3961 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     4021 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     4081 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     4141 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     4201 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     4261 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     4321 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     4381 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     4441 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     4501 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     4561 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     4621 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     4681 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     4741 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     4801 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     4861 gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc
     4921 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     4981 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     5041 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     5101 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     5161 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     5221 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     5281 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     5341 gcgtcccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct
     5401 tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg
     5461 ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgaattgta atacgactca
     5521 ctatagggcg aattggagct ccaccgcggt ggcggccgct ctagaactag tggatccccc
     5581 gggctgcagg aattaattcg tcgacctcga aattctaccg ggtaggggag gcgcttttcc
     5641 caaggcagtc tggagcatgc gctttagcag ccccgctggg cacttggcgc tacacaagtg
     5701 gcctctggcc tcgcacacat tccacatcca ccggtaggcg ccaaccggct ccgttctttg
     5761 gtggcccctt cgcgccacct tctactcctc ccctagtcag gaagttcccc cccgccccgc
     5821 agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg tctcactagt ctcgtgcaga
     5881 tggacagcac cgctgagcaa tggaagcggg taggcctttg gggcagcggc caatagcagc
     5941 tttgctcctt cgctttctgg gctcagaggc tgggaagggg tgggtccggg ggcgggctca
     6001 ggggcgggct caggggcggg gcgggcgccc gaaggtcctc cggaggcccg gcattctgca
     6061 cgcttcaaaa gcgcacgtct gccgcgctgt tctcctcttc ctcatctccg ggcctttcga
     6121 cctgcatcca tctagatctc gatcgagcag ctgaagctta tggagaagtt actattccga
     6181 agttcctatt ctctagaaag tataggaact
//
