LOCUS       sequence-255800-        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_3K
FEATURES             Location/Qualifiers
     source          1..7515
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    586..604
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    607..625
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     635..659
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      819..828
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(825..827,828..830,831..1541)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(825..827,828..830,831..1541)
                     /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(870..891)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1131..1150)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1478..1499
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3233..3250)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3239..3463
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3596..3615)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3747..3794
                     /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             3802..4824
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3802..4824
                     /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    4954..5075
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4991..5010)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5045..5064
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5124..5140)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5124..5140)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5137..5159)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5148..5164
                     /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(5172..5178,5179..5196,5197..5202))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5217..5238
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5355..5372)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5526..6114)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5606..5625)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6285..7076,7077..7145))
                     /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     6908..6927
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7146..7250)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7325..7344)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctccct atcagtgata
      601 gagatctccc tatcagtgat agagatcgtc gacgagctcg tttagtgaac cgtcagatcg
      661 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc
      721 tccggactct agcgtttaaa cttaagcttg gtaccgagct cggatccact agtccagtgt
      781 ggtggaattc tgcagatatc cagcacagtg gcggccgcgc caccatggtg agcaagggcg
      841 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc
      901 acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga
      961 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga
     1021 cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca
     1081 agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca
     1141 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc
     1201 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact
     1261 acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact
     1321 tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga
     1381 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt
     1441 ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga
     1501 ccgccgccgg gatcactctc ggcatggacg agctgtacaa gggatccccc gggctgcagg
     1561 aattcaggaa cggggaggcc atctgcagcg ccctgcccac cattccctac cacaaactcg
     1621 ccgacctgcg ctacctgagc cgcggcgcct ctggcactgt gtcgtccgcc cgccacgcag
     1681 actggcgcgt ccaggtggcc gtgaagcacc tgcacatcca cactccgctg ctcgacagtg
     1741 aaagaaagga tgtcttaaga gaagctgaaa ttttacacaa agctagattt agttacattc
     1801 ttccaatttt gggaatttgc aatgagcctg aatttttggg aatagttact gaatacatgc
     1861 caaatggatc attaaatgaa ctcctacata ggaaaactga atatcctgat gttgcttggc
     1921 cattgagatt tcgcatcctg catgaaattg cccttggtgt aaattacctg cacaatatga
     1981 ctcctccttt acttcatcat gacttgaaga ctcagaatat cttattggac aatgaatttc
     2041 atgttaagat tgcagatttt ggtttatcaa agtggcgcat gatgtccctc tcacagtcac
     2101 gaagtagcaa atctgcacca gaaggaggga caattatcta tatgccacct gaaaactatg
     2161 aacctggaca aaaatcaagg gccagtatca gacacgatat atatagctat gcagttatca
     2221 catgggaagt gttatccaga aaacagcctt ttgaagatgt caccaatcct ttgcagataa
     2281 tgtatagtgt gtcacaagga catcgacctg ttattaatga agaaagtttg ccatatgata
     2341 tacctcaccg agcacgtatg atctctctaa tagaaagtgg atgggcacaa aatccagatg
     2401 aaagaccatc tttcttaaaa tgtttaatag aacttgaacc agttttgaga acatttgaag
     2461 agataacttt tcttgaagct gttattcagc taaagaaaac aaagttacag agtgtttcaa
     2521 gtgccattca cctatgtgac aagaagaaaa tggaattatc tctgaacata cctgtaaatc
     2581 atggtccaca agaggaatca tgtggatcct ctcagctcca tgaaaatagt ggttctcctg
     2641 aaacttcaag gtccctgcca gctcctcaag acaatgattt tttatctaga aaagctcaag
     2701 actgttattt tatgaagctg catcactgtc ctggaaatca cagttgggat agcaccattt
     2761 ctggatctca aagggctgca ttctgtgatc acagaaccac tccatgctct tcagcaataa
     2821 taaatccact ctcaactgca ggaaactcag aacgtctgca gcctggtata gcccagcagt
     2881 ggatccagag caaaagggaa gacattgtga accaaatgac agaagcctgc cttaaccagt
     2941 cgctagatgc ccttctgtcc agggacttga tcatgaaaga ggactatgaa cttgttagta
     3001 ccaagcctac aaggacctca aaagtcagac aattactaga cactactgac atccaaggag
     3061 aagaatttgc caaagttata gtacaaaaat tgaaagataa caaacaaatg ggtcttcagc
     3121 cttacccgga aatacttgtg gtttctagat caccatcttt aaatttactt caaaatagaa
     3181 gcatgtaagt gactgctcga gtctagaggg cccgtttaaa cccgctgatc agcctcgact
     3241 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     3301 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     3361 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     3421 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     3481 accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg
     3541 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     3601 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     3661 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     3721 gattagggtg atggttcacg tacctagaag ttcctattcc gaagttccta ttctctagaa
     3781 agtataggaa cttccttggc caaaaagcct gaactcaccg cgacgtctgt cgagaagttt
     3841 ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct
     3901 cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc
     3961 gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt
     4021 ccggaagtgc ttgacattgg ggaattcagc gagagcctga cctattgcat ctcccgccgt
     4081 gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctgcagccg
     4141 gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc
     4201 ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt
     4261 gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc
     4321 gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc
     4381 gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc
     4441 attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc
     4501 tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg
     4561 gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct tgaccaactc
     4621 tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac
     4681 gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg
     4741 gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc
     4801 actcgtccga gggcaaagga atagcacgta ctacgagatt tcgattccac cgccgccttc
     4861 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     4921 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
     4981 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     5041 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     5101 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     5161 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     5221 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     5281 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     5341 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     5401 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     5461 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     5521 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     5581 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     5641 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     5701 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     5761 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     5821 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     5881 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     5941 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     6001 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     6061 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     6121 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     6181 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     6241 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     6301 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     6361 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     6421 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     6481 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     6541 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     6601 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     6661 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     6721 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     6781 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     6841 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     6901 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     6961 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     7021 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     7081 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     7141 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     7201 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     7261 cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc
     7321 tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt
     7381 tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg
     7441 acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg
     7501 ccagatatac gcgtt
//
