LOCUS       sequence-255803-        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
FEATURES             Location/Qualifiers
     source          1..7515
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(146..165)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     493..514
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2248..2265)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2254..2478
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2611..2630)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2762..2809
                     /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             2817..3839
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2817..3839
                     /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    3969..4090
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4006..4025)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4060..4079
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4139..4155)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4139..4155)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4152..4174)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4163..4179
                     /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(4187..4193,4194..4211,4212..4217))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4232..4253
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4370..4387)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4541..5129)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4621..4640)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5300..6091,6092..6160))
                     /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     5923..5942
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6161..6265)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6340..6359)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6531..6910
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6911..7114
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7065..7085
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    7116..7134
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7137..7155
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     7165..7189
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      7349..7358
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(7355..7357,7358..7360,7361..7515,1..556)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(7355..7357,7358..7360,7361..7515,1..556)
                     /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(7400..7421)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
ORIGIN
        1 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
       61 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
      121 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
      181 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
      241 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
      301 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
      361 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
      421 gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga
      481 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
      541 ggacgagctg tacaagggat cccccgggct gcaggaattc aggaacgggg aggccatctg
      601 cagcgccctg cccaccattc cctaccacaa actcgccgac ctgcgctacc tgagccgcgg
      661 cgcctctggc actgtgtcgt ccgcccgcca cgcagactgg cgcgtccagg tggccgtgaa
      721 gcacctgcac atccacactc cgctgctcga cagtgaaaga aaggatgtct taagagaagc
      781 tgaaatttta cacaaagcta gatttagtta cattcttcca attttgggaa tttgcaatga
      841 gcctgaattt ttgggaatag ttactgaata catgccaaat ggatcattaa atgaactcct
      901 acataggaaa actgaatatc ctgatgttgc ttggccattg agatttcgca tcctgcatga
      961 aattgccctt ggtgtaaatt acctgcacaa tatgactcct cctttacttc atcatgactt
     1021 gaagactcag aatatcttat tggacaatga atttcatgtt aagattgcag attttggttt
     1081 atcaaagtgg cgcatgatgt ccctctcaca gtcacgaagt agcaaatctg caccagaagg
     1141 agggacaatt atctatatgc cacctgaaaa ctatgaacct ggacaaaaat caagggccag
     1201 tatcaagcac gatatatata gctatgcagt tatcacatgg gaagtgttat ccagaaaaca
     1261 gccttttgaa gatgtcacca atcctttgca gataatgtat agtgtgtcac aaggacatcg
     1321 acctgttatt aatgaagaaa gtttgccata tgatatacct caccgagcac gtatgatctc
     1381 tctaatagaa agtggatggg cacaaaatcc agatgaaaga ccatctttct taaaatgttt
     1441 aatagaactt gaaccagttt tgagaacatt tgaagagata acttttcttg aagctgttat
     1501 tcagctaaag aaaacaaagt tacagagtgt ttcaagtgcc attcacctat gtgacaagaa
     1561 gaaaatggaa ttatctctga acatacctgt aaatcatggt ccacaagagg aatcatgtgg
     1621 atcctctcag ctccatgaaa atagtggttc tcctgaaact tcaaggtccc tgccagctcc
     1681 tcaagacaat gattttttat ctagaaaagc tcaagactgt tattttatga agctgcatca
     1741 ctgtcctgga aatcacagtt gggatagcac catttctgga tctcaaaggg ctgcattctg
     1801 tgatcacaag accactccat gctcttcagc aataataaat ccactctcaa ctgcaggaaa
     1861 ctcagaacgt ctgcagcctg gtatagccca gcagtggatc cagagcaaaa gggaagacat
     1921 tgtgaaccaa atgacagaag cctgccttaa ccagtcgcta gatgcccttc tgtccaggga
     1981 cttgatcatg aaagaggact atgaacttgt tagtaccaag cctacaagga cctcaaaagt
     2041 cagacaatta ctagacacta ctgacatcca aggagaagaa tttgccaaag ttatagtaca
     2101 aaaattgaaa gataacaaac aaatgggtct tcagccttac ccggaaatac ttgtggtttc
     2161 tagatcacca tctttaaatt tacttcaaaa taaaagcatg taagtgactg ctcgagtcta
     2221 gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     2281 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     2341 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     2401 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     2461 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     2521 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     2581 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     2641 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     2701 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtacct
     2761 agaagttcct attccgaagt tcctattctc tagaaagtat aggaacttcc ttggccaaaa
     2821 agcctgaact caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct
     2881 ccgacctgat gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag
     2941 ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg
     3001 tttatcggca ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggaat
     3061 tcagcgagag cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc
     3121 tgcctgaaac cgaactgccc gctgttctgc agccggtcgc ggaggccatg gatgcgatcg
     3181 ctgcggccga tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc
     3241 aatacactac atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc
     3301 aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc
     3361 tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca
     3421 atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg
     3481 gggattccca atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg
     3541 agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc
     3601 gggcgtatat gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt
     3661 tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga
     3721 ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag
     3781 aagtactcgc cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaatagc
     3841 acgtactacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     3901 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     3961 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     4021 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     4081 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     4141 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     4201 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     4261 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4321 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     4381 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4441 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4501 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4561 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4621 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4681 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     4741 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4801 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4861 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4921 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4981 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     5041 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     5101 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     5161 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     5221 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     5281 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     5341 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     5401 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     5461 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     5521 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5581 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     5641 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     5701 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     5761 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     5821 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     5881 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     5941 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     6001 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     6061 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     6121 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     6181 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     6241 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg
     6301 gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc
     6361 atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag
     6421 caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag
     6481 ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt
     6541 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
     6601 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
     6661 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
     6721 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
     6781 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
     6841 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
     6901 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
     6961 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
     7021 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
     7081 gcgtgtacgg tgggaggtct atataagcag agctctccct atcagtgata gagatctccc
     7141 tatcagtgat agagatcgtc gacgagctcg tttagtgaac cgtcagatcg cctggagacg
     7201 ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc tccggactct
     7261 agcgtttaaa cttaagcttg gtaccgagct cggatccact agtccagtgt ggtggaattc
     7321 tgcagatatc cagcacagtg gcggccgcgc caccatggtg agcaagggcg aggagctgtt
     7381 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     7441 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     7501 caccaccggc aagct
//
