LOCUS       sequence-325436-        6225 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Introduce Dox-inducible (TRE promoter) GFP cDNA plus miR30-embedded 
            shRenilla into (ES) cells by recombination-mediated cassette 
            exchange.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6225)
  TITLE     Karreth lab plasmids
REFERENCE   2  (bases 1 to 6225)
  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_shRen
FEATURES             Location/Qualifiers
     source          1..6225
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     36..55
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     protein_bind    149..196
                     /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)."
     misc_feature    join(219..284,285..322)
                     /label=Ad SA
                     /note="adenovirus major late transcript splice acceptor 
                     (Friedrich and Soriano, 1991)"
     polyA_signal    491..625
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    745..879
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    join(891..909,910..932,933..951,952..974,975..993,994..1016,
                     1017..1035,1036..1058,1059..1077,1078..1100,1101..1119,
                     1120..1142,1143..1161)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     promoter        1194..1232
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1229..1253
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(1338..1340,1341..1343,1344..2057)
                     /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(1383..1404)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1644..1663)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1991..2012
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     ncRNA           2251..2265
                     /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          2448..3020
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     rep_origin      complement(3348..3936)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3428..3447)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4107..4898,4899..4967))
                     /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     4730..4749
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4968..5072)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5098..5553)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5235..5256)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5447..5466
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5680..5702
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5694..5711
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5695..5711
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5718..5737
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5718..5736
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     5769..5785
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5769..5785
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        join(5829..6225,1..103)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(5864..5887)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
ORIGIN
        1 cggggcgggc gcccgaaggt cctccggagg cccggcattc tgcacgcttc aaaagcgcac
       61 gtctgccgcg ctgttctcct cttcctcatc tccgggcctt tcgacctgca tccatctaga
      121 tctcgatcga gcagctgaag cttatggaga agttactatt ccgaagttcc tattctctag
      181 aaagtatagg aacttcgatc ccccgggctg cagatctgta gggcgcagta gtccagggtt
      241 tccttgatga tgtcatactt atcctgtccc ttttttttcc acagctcgcg gttgaggaca
      301 aactcttcgc ggtctttcca gtggggatcg acggtatcga taagcttgca gatctgcgac
      361 tctagaggat ctgcgactct agaggatcat aatcagccat accacatttg tagaggtttt
      421 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
      481 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
      541 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
      601 caatgtatct tatcatgtct ggatctgcga ctctagagga tcataatcag ccataccaca
      661 tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat
      721 aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa
      781 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
      841 ttgtccaaac tcatcaatgt atcttatcat gtctggatcg agtttaccac tccctatcag
      901 tgatagagaa aagtgaaagt cgagtttacc actccctatc agtgatagag aaaagtgaaa
      961 gtcgagttta ccactcccta tcagtgatag agaaaagtga aagtcgagtt taccactccc
     1021 tatcagtgat agagaaaagt gaaagtcgag tttaccactc cctatcagtg atagagaaaa
     1081 gtgaaagtcg agtttaccac tccctatcag tgatagagaa aagtgaaagt cgagtttacc
     1141 actccctatc agtgatagag aaaagtgaaa gtcgagctcg gtacccgggt cgaggtaggc
     1201 gtgtacggtg ggaggcctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga
     1261 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgcg
     1321 gccccgaatt gaccaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     1381 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtct ggcgagggcg
     1441 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     1501 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
     1561 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     1621 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     1681 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     1741 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
     1801 ccgacaagca gaagaacggc atcaaggcga acctcaagat ccgccacaac atcgaggacg
     1861 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     1921 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
     1981 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     2041 acgagctgta caagtaatga cgtacggcgg ccgcctcgac tagggataac agggtaattg
     2101 tttgaatgag gcttcagtac tttacagaat cgttgcctgc acatcttgga aacacttgct
     2161 gggattactt cgacttctta acccaacaga aggctcgaga aggtatattg ctgttgacag
     2221 tgagcgcagg aattataatg cttatctata gtgaagccac agatgtatag ataagcatta
     2281 taattcctat gcctactgcc tcggacttca aggggctaga attcgagcaa ttatcttgtt
     2341 tactaaaact gaataccttg ctatctcttt gatacatttt tacaaagctg aattaaaatg
     2401 gtataaatta aatcactttt ttcaattgac gcgttgagaa cttcagggtg agtttgggga
     2461 cccttgattg ttctttcttt ttcgctattg taaaattcat gttatatgga gggggcaaag
     2521 ttttcagggt gttgtttaga atgggaagat gtcccttgta tcaccatgga ccctcatgat
     2581 aattttgttt ctttcacttt ctactctgtt gacaaccatt gtctcctctt attttctttt
     2641 cattttctgt aactttttcg ttaaacttta gcttgcattt gtaacgaatt tttaaattca
     2701 cttttgttta tttgtcagat tgtaagtact ttctctaatc actttttttt caaggcaatc
     2761 agggtatatt atattgtact tcagcacagt tttagagaac aattgttata attaaatgat
     2821 aaggtagaat atttctgcat ataaattctg gctggcgtgg aaatattctt attggtagaa
     2881 acaactacac cctggtcatc atcctgcctt tctctttatg gttacaatga tatacactgt
     2941 ttgagatgag gataaaatac tctgagtcca aaccgggccc ctctgctaac catgttcatg
     3001 ccttcttctc tttcctacag ctcctgggca acgtgctggt tgttgtgctg tctcatcatt
     3061 ttggcaaagg attcactcct caggtgcagg ctgcctatca gaaggtggtg gctggtgtgg
     3121 ccaatgccct ggctcacaaa taccactgag atcgttttcc ctctgccaaa aattatgggg
     3181 acatcatgaa gccccttgag catctgactt ctggctaata aaggaaattt attttcattg
     3241 caatagtgtg ttggaatttc tcgatcgcta gcatcctagg cgcgccacat gtgagcaaaa
     3301 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     3361 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     3421 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     3481 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     3541 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     3601 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     3661 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     3721 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     3781 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     3841 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     3901 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     3961 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     4021 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     4081 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     4141 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     4201 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     4261 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     4321 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     4381 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     4441 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     4501 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     4561 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     4621 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     4681 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     4741 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     4801 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     4861 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     4921 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     4981 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     5041 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa
     5101 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     5161 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     5221 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     5281 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     5341 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     5401 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     5461 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     5521 ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccattcagg ctgcgcaact
     5581 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
     5641 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa
     5701 cgacggccag tgaattgtaa tacgactcac tatagggcga attggagctc caccgcggtg
     5761 gcggccgctc tagaactagt ggatcccccg ggctgcagga attaattcgt cgacctcgaa
     5821 attctaccgg gtaggggagg cgcttttccc aaggcagtct ggagcatgcg ctttagcagc
     5881 cccgctgggc acttggcgct acacaagtgg cctctggcct cgcacacatt ccacatccac
     5941 cggtaggcgc caaccggctc cgttctttgg tggccccttc gcgccacctt ctactcctcc
     6001 cctagtcagg aagttccccc ccgccccgca gctcgcgtcg tgcaggacgt gacaaatgga
     6061 agtagcacgt ctcactagtc tcgtgcagat ggacagcacc gctgagcaat ggaagcgggt
     6121 aggcctttgg ggcagcggcc aatagcagct ttgctccttc gctttctggg ctcagaggct
     6181 gggaaggggt gggtccgggg gcgggctcag gggcgggctc agggg
//
