LOCUS       sequence-352112-        7850 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  construct expresses tet repressor (tetR) to reduce leakiness 
            together with the fluorochrome t-Sapphire.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7850)
  AUTHORS   Liu WH, Mrozek-Gorska P, Wirth AK, Herold T, Schwarzkopf L, Pich D, 
            Volse K, Melo-Narvaez MC, Carlet M, Hammerschmidt W, Jeremias I
  TITLE     Inducible transgene expression in PDX models in vivo identifies KLF4
            as a therapeutic target for B-ALL.
  JOURNAL   Biomark Res. 2020 Sep 16;8:46. doi: 10.1186/s40364-020-00226-z. 
            eCollection 2020.
  PUBMED    32944247
REFERENCE   2  (bases 1 to 7850)
  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: "Biomark 
            Res."; date: "2020-09-16"; volume: "8:46. doi"; pages: " 
            10.1186/s40364-020-00226-z. eCollection 2020"
COMMENT     Sequence Label: pCDH-EF1a-tetR-T2A-tSapphire
FEATURES             Location/Qualifiers
     source          1..7850
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        6..232
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             233..413
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    460..585
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1078..1311
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1496..1540
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    1807..1923
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1966..2177
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             2190..2458
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             2504..3124
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(2577..2596)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             3164..3217
                     /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(3224..3226,3227..3229,3230..3940)
                     /codon_start=1
                     /product="strongly enhanced green fluorescent protein 
                     (Kremers et al., 2007)"
                     /label=SGFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFSYGVMVFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSIQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3269..3290)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3530..3549)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3877..3898
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3950..4538
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4003..4023)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4421..4432)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4612..4845
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4917..5038
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4954..4973)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5008..5027
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5057..5192
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5225..5241)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5225..5241)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5238..5260)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5249..5265
                     /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(5273..5279,5280..5297,5298..5303))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5318..5339
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5456..5473)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5627..6215)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(6386..7177,7178..7246))
                     /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     7009..7028
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7247..7351)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7419..7437
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7475..7497)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7597..7616
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7810..7832
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7824..7841
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7825..7841
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 acgcgtgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca
       61 acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta
      121 cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga
      181 attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacataa acgggtctct
      241 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa
      301 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc
      361 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc
      421 ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg
      481 cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt
      541 ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg
      601 agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa
      661 ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg
      721 ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca
      781 ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa
      841 aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa
      901 agtaagacca ccgcacagca agcggccact gatcttcaga cctggaggag gagatatgag
      961 ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt
     1021 agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg
     1081 agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac
     1141 gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct
     1201 gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct
     1261 ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg
     1321 gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa
     1381 taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa
     1441 caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa
     1501 tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac
     1561 aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag
     1621 aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc
     1681 gtttcagacc cacctcccaa ccccgagggg acccgacagg cccgaaggaa tagaagaaga
     1741 aggtggagag agagacagag acagatccat tcgattagtg aacggatctc gacggtatcg
     1801 gttaactttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga
     1861 cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa aattcaaaat
     1921 tttatcgata ctagtggatc tgcgatcgct ccggtgcccg tcagtgggca gagcgcacat
     1981 cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaacgggt gcctagagaa
     2041 ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg
     2101 gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt
     2161 ttgccgccag aacacagctg aagcttcgag gggctcgcat ctctccttca cgcgcccgcc
     2221 gccctacctg aggccgccat ccacgccggt tgagtcgcgt tctgccgcct cccgcctgtg
     2281 gtgcctcctg aactgcgtcc gccgtctagg taagtttaaa gctcaggtcg agaccgggcc
     2341 tttgtccggc gctcccttgg agcctaccta gactcagccg gctctccacg ctttgcctga
     2401 ccctgcttgc tcaactctac gtctttgttt cgttttctgt tctgcgccgt tacagatcca
     2461 agctgtgacc ggcgcctact ctagagctag cgaattcgcc accatgtcta gattagataa
     2521 aagtaaagtg attaacagcg cattagagct gcttaatgag gtcggaatcg aaggtttaac
     2581 aacccgtaaa ctcgcccaga agctaggtgt agagcagcct acattgtatt ggcatgtaaa
     2641 aaataagcgg gctttgctcg acgccttagc cattgagatg ttagataggc accatactca
     2701 cttttgccct ttagaagggg aaagctggca agatttttta cgtaataacg ctaaaagttt
     2761 tagatgtgct ttactaagtc atcgcgatgg agcaaaagta catttaggta cacggcctac
     2821 agaaaaacag tatgaaactc tcgaaaatca attagccttt ttatgccaac aaggtttttc
     2881 actagagaat gcattatatg cactcagcgc tgtggggcat tttactttag gttgcgtatt
     2941 ggaagatcaa gagcatcaag tcgctaaaga agaaagggaa acacctacta ctgatagtat
     3001 gccgccatta ttacgacaag ctatcgaatt atttgatcac caaggtgcag agccagcctt
     3061 cttattcggc cttgaattga tcatatgcgg attagaaaaa caacttaaat gtgaaagtgg
     3121 gtccgcgtac agcggatccc gggaattcgg atccgcggcc gctgagggca gaggaagtct
     3181 tctaacatgc ggtgacgtgg aggagaatcc cggccctatg catatggtga gcaagggcga
     3241 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
     3301 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa
     3361 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccttctc
     3421 ctacggcgtg atggtgttcg cccgctaccc cgaccacatg aagcagcacg acttcttcaa
     3481 gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa
     3541 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
     3601 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaactt
     3661 caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggccaactt
     3721 caagatccgc cacaacatcg aggacggcgg cgtgcagctc gccgaccact accagcagaa
     3781 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcatccagtc
     3841 caagctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
     3901 cgccgccggg atcactctcg gcatggacga gctgtacaag taagtcgaca atcaacctct
     3961 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
     4021 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
     4081 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt
     4141 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat
     4201 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc
     4261 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
     4321 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc
     4381 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga
     4441 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc
     4501 tcagacgagt cggatctccc tttgggccgc ctccccgcct ggtaccttta agaccaatga
     4561 cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc
     4621 taattcactc ccaacgaaga taagatctgc tttttgcttg tactgggtct ctctggttag
     4681 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
     4741 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
     4801 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtagt agttcatgtc
     4861 atcttattat tcagtattta taacttgcaa agaaatgaat atcagagagt gagaggaact
     4921 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     4981 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     5041 atgtctggct ctagctatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     5101 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt
     5161 ccagaagtag tgaggaggct tttttggagg cctagacttt tgcagagacc aaattcgtaa
     5221 tcatgtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     5281 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     5341 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     5401 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     5461 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     5521 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     5581 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     5641 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5701 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5761 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5821 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5881 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5941 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     6001 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     6061 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     6121 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     6181 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     6241 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     6301 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     6361 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     6421 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     6481 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     6541 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     6601 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     6661 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     6721 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6781 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6841 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     6901 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     6961 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     7021 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     7081 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     7141 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     7201 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     7261 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     7321 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     7381 tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct
     7441 ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
     7501 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
     7561 cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga
     7621 gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
     7681 ggcgccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt
     7741 cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc
     7801 cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgccaagctg
//
