LOCUS       sequence-255729-        7515 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression, tet R.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7515)
  AUTHORS   Ellwanger K, Briese S, Arnold C, Kienes I, Heim V, Nachbur U, Kufer 
            TA
  TITLE     XIAP controls RIPK2 signaling by preventing its deposition in 
            speck-like structures.
  JOURNAL   Life Sci Alliance. 2019 Jul 26;2(4). pii: 2/4/e201900346. doi: 
            10.26508/lsa.201900346. Print 2019 Aug.
  PUBMED    31350258
REFERENCE   2  (bases 1 to 7515)
  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: "Life Sci 
            Alliance."; date: "2019-07-26"; volume: "2(4). pii: 2/4/e201900346. 
            doi"; pages: " 10.26508/lsa.201900346. Print 2019 Aug"
COMMENT     Sequence Label: pcDNA5-FRT-TO-EGFP-RIP2_Y474F
FEATURES             Location/Qualifiers
     source          1..7515
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    206..224
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    227..245
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     255..279
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      439..448
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(445..447,448..450,451..1161)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(445..447,448..450,451..1161)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(490..511)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(751..770)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1098..1119
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2853..2870)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2859..3083
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3216..3235)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3367..3414
                     /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)."
     CDS             3422..4444
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3422..4444
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4574..4695
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4611..4630)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4665..4684
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4744..4760)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4744..4760)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4757..4779)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4768..4784
                     /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(4792..4798,4799..4816,4817..4822))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4837..4858
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4975..4992)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5146..5734)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5226..5245)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5905..6696,6697..6765))
                     /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     6528..6547
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6766..6870)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6945..6964)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        7136..7515
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctctccct atcagtgata gagatctccc tatcagtgat
      241 agagatcgtc gacgagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc
      301 tgttttgacc tccatagaag acaccgggac cgatccagcc tccggactct agcgtttaaa
      361 cttaagcttg gtaccgagct cggatccact agtccagtgt ggtggaattc tgcagatatc
      421 cagcacagtg gcggccgcgc caccatggtg agcaagggcg aggagctgtt caccggggtg
      481 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
      541 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
      601 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
      661 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
      721 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
      781 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
      841 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
      901 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc
      961 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     1021 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc
     1081 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     1141 ggcatggacg agctgtacaa gggatccccc gggctgcagg aattcaggaa cggggaggcc
     1201 atctgcagcg ccctgcccac cattccctac cacaaactcg ccgacctgcg ctacctgagc
     1261 cgcggcgcct ctggcactgt gtcgtccgcc cgccacgcag actggcgcgt ccaggtggcc
     1321 gtgaagcacc tgcacatcca cactccgctg ctcgacagtg aaagaaagga tgtcttaaga
     1381 gaagctgaaa ttttacacaa agctagattt agttacattc ttccaatttt gggaatttgc
     1441 aatgagcctg aatttttggg aatagttact gaatacatgc caaatggatc attaaatgaa
     1501 ctcctacata ggaaaactga atatcctgat gttgcttggc cattgagatt tcgcatcctg
     1561 catgaaattg cccttggtgt aaattacctg cacaatatga ctcctccttt acttcatcat
     1621 gacttgaaga ctcagaatat cttattggac aatgaatttc atgttaagat tgcagatttt
     1681 ggtttatcaa agtggcgcat gatgtccctc tcacagtcac gaagtagcaa atctgcacca
     1741 gaaggaggga caattatcta tatgccacct gaaaactatg aacctggaca aaaatcaagg
     1801 gccagtatca agcacgatat atatagctat gcagttatca catgggaagt gttatccaga
     1861 aaacagcctt ttgaagatgt caccaatcct ttgcagataa tgtatagtgt gtcacaagga
     1921 catcgacctg ttattaatga agaaagtttg ccatatgata tacctcaccg agcacgtatg
     1981 atctctctaa tagaaagtgg atgggcacaa aatccagatg aaagaccatc tttcttaaaa
     2041 tgtttaatag aacttgaacc agttttgaga acatttgaag agataacttt tcttgaagct
     2101 gttattcagc taaagaaaac aaagttacag agtgtttcaa gtgccattca cctatgtgac
     2161 aagaagaaaa tggaattatc tctgaacata cctgtaaatc atggtccaca agaggaatca
     2221 tgtggatcct ctcagctcca tgaaaatagt ggttctcctg aaacttcaag gtccctgcca
     2281 gctcctcaag acaatgattt tttatctaga aaagctcaag actgttattt tatgaagctg
     2341 catcactgtc ctggaaatca cagttgggat agcaccattt ctggatctca aagggctgca
     2401 ttctgtgatc acaagaccac tccatgctct tcagcaataa taaatccact ctcaactgca
     2461 ggaaactcag aacgtctgca gcctggtata gcccagcagt ggatccagag caaaagggaa
     2521 gacattgtga accaaatgac agaagcctgc cttaaccagt cgctagatgc ccttctgtcc
     2581 agggacttga tcatgaaaga ggactttgaa cttgttagta ccaagcctac aaggacctca
     2641 aaagtcagac aattactaga cactactgac atccaaggag aagaatttgc caaagttata
     2701 gtacaaaaat tgaaagataa caaacaaatg ggtcttcagc cttacccgga aatacttgtg
     2761 gtttctagat caccatcttt aaatttactt caaaataaaa gcatgtaagt gactgctcga
     2821 gtctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc
     2881 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     2941 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     3001 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     3061 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg
     3121 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     3181 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     3241 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     3301 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     3361 tacctagaag ttcctattcc gaagttccta ttctctagaa agtataggaa cttccttggc
     3421 caaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag
     3481 cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt
     3541 aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg
     3601 ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg
     3661 ggaattcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca
     3721 agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc
     3781 gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat
     3841 cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca
     3901 ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct
     3961 gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc
     4021 caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat
     4081 gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg
     4141 tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg
     4201 gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg
     4261 caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc
     4321 cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg
     4381 tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga
     4441 atagcacgta ctacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     4501 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     4561 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     4621 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     4681 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc
     4741 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     4801 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     4861 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4921 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     4981 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     5041 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     5101 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     5161 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     5221 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     5281 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     5341 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     5401 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5461 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     5521 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5581 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     5641 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     5701 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     5761 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     5821 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     5881 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     5941 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     6001 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     6061 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     6121 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     6181 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     6241 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     6301 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     6361 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     6421 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     6481 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     6541 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     6601 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     6661 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     6721 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     6781 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     6841 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     6901 cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat
     6961 gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc
     7021 gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg
     7081 cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat
     7141 tgattattga ctagttatta atagtaatca attacggggt cattagttca tagcccatat
     7201 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
     7261 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc
     7321 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
     7381 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
     7441 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
     7501 atcgctatta ccatg
//
