LOCUS       sequence-476965-        5007 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid expressing a rapamycin-inducible transcriptional activator.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5007)
  AUTHORS   Peterman EL, Ploessl DS, Love KS, Sanabria V, Daniels RF, Johnstone 
            CP, Godavarti DR, Kabaria SR, Oakes CG, Pai AA, Galloway KE
  TITLE     High-resolution profiling reveals coupled transcriptional and 
            translational regulation of transgenes.
  JOURNAL   Nucleic Acids Research, Volume 53, Issue 11, 24 June 2025, gkaf528
  PUBMED    39651241
REFERENCE   2  (bases 1 to 5007)
  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: "Nucleic 
            Acids Research, Volume 53, Issue 11, 24 June 2025, gkaf528"
COMMENT     Sequence Label: 
            pKG3666_pGEEC576_pShip-EFS-ZF43a(inducible)-P2A-iRFP720-bGH
FEATURES             Location/Qualifiers
     source          1..5007
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        5..216
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      222..231
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             231..296
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             303..323
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(366..368,369..689)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     CDS             1026..1082
                     /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             1170..1319
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             1722..1778
                     /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             1779..2729
                     /codon_start=1
                     /product="near-infrared fluorescent protein with an 
                     emission peak at 720 nm, engineered from a bacterial 
                     phytochrome (Shcherbakova and Verkhusha, 2013)"
                     /label=iRFP720
                     /note="derived from Rhodopseudomonas palustris RpBphP2"
                     /translation="MAEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGS
                     DNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFI
                     GSWHRHDQLIFLELEPPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQEVRKIT
                     GFDRVMIYRFASDFSGSVIAEDRCAEVESKLGLHYPASFIPAQARRLYTINPVRIIPDI
                     NYRPVPVTPDLNPVTGRPIDLSFAILRSVSPNHLEFMRNIGMHGTMSISILRGERLWGL
                     IVCHHRTPYYVDLDGRQACELVAQVLAWQIGVMEE"
     polyA_signal    2741..2965
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3076..3095)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3087..3896
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPGDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3154..3173)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3986..4574
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4475..4494
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      4739..4825
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4917..4944
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     4952..4975
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
ORIGIN
        1 tactgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat
       61 tgaaccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg
      121 ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac
      181 gttctttttc gcaacgggtt tgccgccaga acacagtaat ggccaccatg gattataagg
      241 atcacgatgg cgactacaag gaccacgata ttgactacaa agacgatgac gataaaatgg
      301 ctcctaagaa aaagcgcaaa gtcggtatcc atggcgttcc cgtcgacggt ggactccaag
      361 ttaagatggg agtgcaggtg gaaaccatct ccccaggaga cgggcgcacc ttccccaagc
      421 gcggccagac ctgcgtggtg cactacaccg ggatgcttga agatggaaag aaatttgatt
      481 cctcccggga cagaaacaag ccctttaagt ttatgctagg caagcaggag gtgatccgag
      541 gctgggaaga aggggttgcc cagatgagtg tgggtcagag agccaaactg actatatctc
      601 cagattatgc ctatggtgcc actgggcacc caggcatcat cccaccacat gccactctcg
      661 tctttgatgt ggagcttcta aaactggaag gatccggtgg tggcgggtct ggaggaggag
      721 gtagtggtgg aggtggctct ggtaccgcta gacccgggga gcgccccttc cagtgtcgca
      781 tttgcatgcg gaacttttcg cgccaggaca ggcttgacag gcatacccgt actcataccg
      841 gtgaaaaacc gtttcagtgt cggatctgta tgcgaaattt ctcccagaag gagcacttgg
      901 cggggcatct acgtacgcac accggcgaga agccattcca atgccgaata tgcatgcgca
      961 acttcagtcg ccgcgacaac ctgaaccggc acctaaaaac ccacctgaga ggatctggaa
     1021 gcggagctac taacttcagc ctgctgaagc aggctggaga cgtggaggag aaccctggac
     1081 ctgcaccagg aagtatggag gaactttcac aggccctggc tagtagtttt tcagtgtctg
     1141 gtgtcggtat ccatggcgtt cccggatcag acgcattgga cgattttgat ctggatatgc
     1201 tgggaagtga cgccctcgat gattttgacc ttgacatgct tggttcggat gcccttgatg
     1261 actttgacct cgacatgctc ggcagtgacg cccttgatga tttcgacctg gacatgctga
     1321 ttaactctag aagttccgga tcttctggtt ctggttctgg ttcaggatcc ggtggtggcg
     1381 ggtctggagg aggaggtagt ggtggaggtg gctctggtac catgcttgag atgtggcatg
     1441 aaggcctgga agaggcatct cgtttgtact ttggggaaag gaacgtgaaa ggcatgtttg
     1501 aggtgctgga gcccttgcat gctatgatgg aacggggacc ccagactctg aaggaaacat
     1561 cctttaatca ggcctatggt cgagatttaa tggaggccca agagtggtgc aggaagtaca
     1621 tgaaatcagg gaatgtcaag gacctcctcc aagcctggga cctctattat catgtgttcc
     1681 gacgaatctc aaagactaga gctagcggcc gcggaagcgg agctactaac ttcagcctgc
     1741 tgaagcaggc tggagacgtg gaggagaacc ctggacctat ggcggaagga tccgtcgcca
     1801 ggcagcctga cctcttgacc tgcgacgatg agccgatcca tatccccggt gccatccaac
     1861 cgcatggact gctgctcgcc ctcgccgccg acatgacgat cgttgccggc agcgacaacc
     1921 ttcccgaact caccggactg gcgatcggcg ccctgatcgg ccgctctgcg gccgatgtct
     1981 tcgactcgga gacgcacaac cgtctgacga tcgccttggc cgagcccggg gcggccgtcg
     2041 gagcaccgat cactgtcggc ttcacgatgc gaaaggacgc aggcttcatc ggctcctggc
     2101 atcgccatga tcagctcatc ttcctcgagc tcgagcctcc ccagcgggac gtcgccgagc
     2161 cgcaggcgtt cttccgccgc accaacagcg ccatccgccg cctgcaggcc gccgaaacct
     2221 tggaaagcgc ctgcgccgcc gcggcgcaag aggtgcggaa gattaccggc ttcgatcggg
     2281 tgatgatcta tcgcttcgcc tccgacttca gcgggtccgt gatcgcagag gatcggtgcg
     2341 ccgaggtcga gtcaaaacta ggcctgcact atcctgcctc attcatcccg gcgcaggccc
     2401 gtcggctcta taccatcaac ccggtacgga tcattcccga tatcaattat cggccggtgc
     2461 cggtcacccc agacctcaat ccggtcaccg ggcggccgat tgatcttagc ttcgccatcc
     2521 tgcgcagcgt ctcgcccaac catctggagt tcatgcgcaa cataggcatg cacggcacga
     2581 tgtcgatctc gattttgcgc ggcgagcgac tgtggggatt gatcgtttgc catcaccgaa
     2641 cgccgtacta cgtcgatctc gatggccgcc aagcctgcga gctagtcgcc caggttctgg
     2701 cctggcagat cggcgtgatg gaagagtaac aacgttgcac ctgtgccttc tagttgccag
     2761 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     2821 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     2881 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     2941 gctggggatg cggtgggctc tatggcgctc gctgactgca ggtgcccgtg tctcaaaatc
     3001 tctgatgtta cattgcacaa gataaaaata tatcatcatg aacaataaaa ctgtctgctt
     3061 acataaacag taatacaagg ggtgttatga gccatattca acgggaaacg tcgaggccgc
     3121 gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg
     3181 ggcaatcagg tgcgacaatc tatcgcttgt atgggaagcc cgatgcgcca gagttgtttc
     3241 tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact
     3301 ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact cctggtgatg
     3361 catggttact caccactgcg atccccggaa aaacagcatt ccaggtatta gaagaatatc
     3421 ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga
     3481 ttcctgtttg taattgtcct tttaacagcg atcgcgtatt tcgtctagct caggcgcaat
     3541 cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc
     3601 ctgttgaaca agtctggaaa gaaatgcata aacttttgcc attctcaccg gattcagtcg
     3661 tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt
     3721 gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga
     3781 actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg
     3841 ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaatcag
     3901 aattggttaa ttggttgtaa cattattcag attgggcccc gttccactga gcgtcagacc
     3961 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     4021 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     4081 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     4141 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     4201 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     4261 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     4321 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     4381 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     4441 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     4501 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     4561 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     4621 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     4681 ctagcatgga tctcggggac gtctaactac taagcgagag tagggaactg ccaggcatca
     4741 aataaaacga aaggctcagt cggaagactg ggcctttcgt tttatctgtt gtttgtcggt
     4801 gaacgctctc ctgagtagga caaatccgcc gggagcggat ttgaacgttg tgaagcaacg
     4861 gcccggaggg tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa ctaagcagaa
     4921 ggccatcctg acggatggcc tttttgcgtt tctacaaact cttcctgtta gttagttact
     4981 taagctcggg cccacctgca gtctact
//
