LOCUS       sequence-82852-e        4074 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pEntry Tshld-GFP is a product of BP recombination reaction between 
            attB flanked Tshld system-encoded PCR product and pDonor 201 
            plasmid..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4074)
  AUTHORS   Lin YH, Pratt MR
  TITLE     A Dual Small-Molecule Rheostat for Precise Control of Protein 
            Concentration in Mammalian Cells.
  JOURNAL   Chembiochem. 2014 Mar 11. doi: 10.1002/cbic.201400006.
  PUBMED    24615791
REFERENCE   2  (bases 1 to 4074)
  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: 
            "Chembiochem. 2014 Mar 11. doi: 10.1002/cbic.201400006."
COMMENT     Sequence Label: pEntry Tshld-GFP
FEATURES             Location/Qualifiers
     source          1..4074
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(73..100)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(192..278)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    331..430
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             434..457
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             470..748
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     CDS             920..973
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(983..985,986..1306)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKPE"
     CDS             1490..1516
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(1523..1525,1526..1528,1529..2242)
                     /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(1523..1525,1526..1528,1529..2239)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1568..1589)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1829..1848)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2176..2197
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2250..2349)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2461..2480)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2472..3281
                     /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
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2539..2558)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3424..4012
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3913..3932
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgctagcc aggaagagtt
       61 tgtagaaacg caaaaaggcc atccgtcagg atggccttct gcttagtttg atgcctggca
      121 gtttatggcg ggcgtcctgc ccgccaccct ccgggccgtt gcttcacaac gttcaaatcc
      181 gctcccggcg gatttgtcct actcaggaga gcgttcaccg acaaacaaca gataaaacga
      241 aaggcccagt cttccgactg agcctttcgt tttatttgat gcctggcagt tccctactct
      301 cgcgttaacg ctagcatgga tctcgggccc caaataatga ttttattttg actgatagtg
      361 acctgttcgt tgcaacaaat tgatgagcaa tgctttttta taatgccaac tttgtacaaa
      421 aaagcaggct atggactaca aagacgatga cgataaaatg gcttccagaa tcctctggca
      481 tgagatgtgg catgaaggcc tggaagaggc atctcgtttg tactttgggg aaaggaacgt
      541 gaaaggcatg tttgaggtgc tggagccctt gcatgctatg atggaacggg gcccccagac
      601 tctgaaggaa acatccttta atcaggccta tggtcgagat ttaatggagg cccaagagtg
      661 gtgcaggaag tacatgaaat cagggaatgt caaggacctc ctccaagcct gggacctcta
      721 ttatcatgtg ttccgacgaa tctcaaaggg taccggcggc ggcagcggcg gcggcagcgg
      781 cggcggcagc ggcggcggca gcatgcagat cttcgtgaag accctgactg gtaagaccgg
      841 caccctggag gtggagccca gtgacaccat cgagaatgtc aaggcaaaga tccaagataa
      901 ggaaggcatc cctggcggcg agggcagggg ctccctgctg acctgcggcg acgtggagga
      961 gaaccccggc cccatgctcg agatgggagt gcaggtggaa accatctccc caggagacgg
     1021 gcgcaccttc cccaagcgcg gccagacctg cgtggtgcac tacaccggga tgcttgaaga
     1081 tggaaagaaa tttgattcct cccgggacag aaacaagccc tttaagttta tgctaggcaa
     1141 gcaggaggtg atccgaggct gggaagaagg ggttgcccag atgagtgtgg gtcagagagc
     1201 caaactgact atatctccag attatgccta tggtgccact gggcacccag gcatcatccc
     1261 accacatgcc actctcgtct tcgatgtgga gcttctaaaa ccggaaggta ccggcggcgg
     1321 cagcggcggc ggcagcggcg gcggcagcgg cggcggcagc ggtatccctc cagatcaaca
     1381 aagattgatc tttgccggta agcagctaga agacggtaga acgctgtctg attacaacat
     1441 tcagaaggag tccaccttac atctggtctt gagactccgc ggtggtatgt acccctacga
     1501 cgtgccagac tacgcggaat tcatggtgag caagggcgag gagctgttca ccggggtggt
     1561 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     1621 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     1681 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
     1741 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     1801 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     1861 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     1921 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     1981 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
     2041 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     2101 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa
     2161 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     2221 catggacgag ctgtacaagt aatctagaga cccagctttc ttgtacaaag ttggcattat
     2281 aagaaagcat tgcttatcaa tttgttgcaa cgaacaggtc actatcagtc aaaataaaat
     2341 cattatttgc catccagctg cagctctggc ccgtgtctca aaatctctga tgttacattg
     2401 cacaagataa aaatatatca tcatgaacaa taaaactgtc tgcttacata aacagtaata
     2461 caaggggtgt tatgagccat attcaacggg aaacgtcgag gccgcgatta aattccaaca
     2521 tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga
     2581 caatctatcg cttgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag
     2641 gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta
     2701 tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca
     2761 ctgcgatccc cggaaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa
     2821 atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt
     2881 gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg
     2941 gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct
     3001 ggaaagaaat gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt
     3061 tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac
     3121 gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt
     3181 tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga
     3241 ataaattgca gtttcatttg atgctcgatg agtttttcta atcagaattg gttaattggt
     3301 tgtaacactg gcagagcatt acgctgactt gacgggacgg cgcaagctca tgaccaaaat
     3361 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     3421 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     3481 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     3541 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     3601 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     3661 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     3721 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     3781 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     3841 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     3901 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     3961 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     4021 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgtt
//
