LOCUS       V029771                 8043 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V029771
VERSION     V029771
KEYWORDS    .
SOURCE      .
  ORGANISM  .
            .
FEATURES             Location/Qualifiers
     enhancer        50..429
                     /note="human cytomegalovirus immediate early enhancer"
                     /label="CMV enhancer"
     promoter        430..633
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
                     /label="CMV promoter"
     promoter        678..696
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /label="T7 promoter"
     CDS             756..2723
                     /label="Ripk1(NM_009068)"
                     /note="Ripk1(NM_009068)"
                     /gene="Ripk1"
     CDS             2730..2795
                     /codon_start=1
                     /product="three tandem FLAG® epitope tags, followed by an
                     enterokinase cleavage site"
                     /transl_table=1
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
                     /label="3xFLAG"
     misc_feature    2819..3407
                     /note="woodchuck hepatitis virus&nbsp;posttranscriptional
                     regulatory element"
                     /label="WPRE"
     polyA_signal    3473..3521
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal&nbsp;(Cole and Stacy, 1985)"
                     /label="HSV TK poly(A) signal"
     rep_origin      3723..4151
                     /direction=RIGHT
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     promoter        4165..4494
                     /note="SV40 enhancer and early promoter"
                     /label="SV40 promoter"
     CDS             4561..5355
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /note="confers resistance to neomycin, kanamycin, and G418
                     (Geneticin®)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /transl_table=1
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*"
                     /label="NeoR/KanR"
     polyA_signal    5531..5652
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     primer_bind     complement(5701..5717)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    5725..5741
                     /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-β-D-thiogalactopyranoside (IPTG)."
                     /label="lac operator"
     promoter        complement(5749..5779)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    5794..5815
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(6103..6691)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(6862..7722)
                     /codon_start=1
                     /gene="bla"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /product="β-lactamase"
                     /transl_table=1
                     /translation="MSIQHFRVALIPFFAAFCLPVFA,HPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEY
                     SPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDR
                     WEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRS
                     ALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGA
                     SLIKHW*"
                     /label="AmpR"
     promoter        complement(7723..7827)
                     /gene="bla"
                     /label="AmpR promoter"
ORIGIN
        1 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
       61 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      121 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      181 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      241 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      301 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      361 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      421 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      481 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      541 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      601 cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga acccactgct
      661 tactggctta tcgaaattaa tacgactcac tatagggaga cccaagctgg ctagcgttta
      721 aacttaagct tggtaccgag ctcgaattcg ccaccatgca accagacatg tccttggaca
      781 atattaagat ggcatccagt gacctgctgg agaagacaga cctagacagc ggaggcttcg
      841 ggaaggtgtc cttgtgttac cacagaagcc atggatttgt catcctgaaa aaagtataca
      901 cagggcccaa ccgcgctgag tacaatgagg ttctcttgga agaggggaag atgatgcaca
      961 gactgagaca cagtcgagtg gtgaagctac tgggcatcat catagaagaa gggaactatt
     1021 cgctggtgat ggagtacatg gagaagggca acctgatgca cgtgctaaag acccagatag
     1081 atgtcccact ttcattgaaa ggaaggataa tcgtggaggc catagaaggc atgtgctact
     1141 tacatgacaa aggtgtgata cacaaggacc tgaagcctga gaatatcctc gttgatcgtg
     1201 actttcacat taagatagcc gatcttggtg tggcttcctt taagacatgg agcaaactga
     1261 ctaaggagaa agacaacaag cagaaagaag tgagcagcac cactaagaag aacaatggtg
     1321 gtacccttta ctacatggca cccgaacacc tgaatgacat caatgcaaag cccacggaga
     1381 agtcggacgt gtacagcttt ggcattgtcc tttgggcaat atttgcaaaa aaggagccct
     1441 atgagaatgt catctgtact gagcagttcg tgatctgcat aaaatctggg aacaggccaa
     1501 atgtagagga aatccttgag tactgtccaa gggagatcat cagcctcatg gagcggtgct
     1561 ggcaggcgat cccagaagac aggccaacat ttcttggcat tgaagaagaa tttaggcctt
     1621 tttacttaag tcattttgaa gaatatgtag aagaggatgt ggcaagttta aagaaagagt
     1681 atccagatca aagcccagtg ctgcagagaa tgttttcact gcagcatgac tgtgtaccct
     1741 tacctccgag caggtcaaat tcagaacaac ctggatcgct gcacagttcc caggggctcc
     1801 agatgggtcc tgtggaggag tcctggtttt cttcctcccc agagtaccca caggacgaga
     1861 atgatcgcag tgtgcaggct aagctgcaag aggaagccag ctatcatgct tttggaatat
     1921 ttgcagagaa acagacaaaa ccgcagccaa ggcagaatga ggcttacaac agagaggagg
     1981 aaaggaaacg aagggtctct catgacccct ttgcacagca gagagctcgt gagaatatta
     2041 agagtgcagg agcaagaggt cattctgatc ccagcacaac gagtcgtgga attgcagtgc
     2101 aacagctgtc atggccagcc acccaaacag tttggaacaa tggattgtat aatcagcatg
     2161 gatttggaac tacaggtaca ggagtttggt atccgccaaa tctaagccaa atgtatagta
     2221 cttataaaac tccagtgcca gagaccaaca taccgggaag cacacccacc atgccatact
     2281 tctctgggcc agtagcagat gacctcataa aatatactat attcaatagt tctggtattc
     2341 agattggaaa ccacaattat atggatgttg gactgaattc acaaccacca aacaatactt
     2401 gcaaagaaga gtcgacttcc agacaccaag ccatctttga taacaccact agtctgactg
     2461 atgaacacct gaaccctatc agggaaaacc tgggaaggca gtggaaaaac tgtgcccgca
     2521 agctgggctt cactgagtct cagatcgatg aaatcgacca tgactatgaa agagatggac
     2581 tgaaagagaa agtttaccaa atgcttcaga agtggctgat gcgggaaggc accaaagggg
     2641 ccacagtggg aaagttggcc caggcacttc accaatgttg caggatagac ctgctgaacc
     2701 acttgattcg tgccagccag agcggatccg actacaaaga ccatgacggt gattataaag
     2761 atcatgacat cgactacaag gatgacgatg acaagtagtg agggcccgat atctcgacaa
     2821 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
     2881 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
     2941 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg
     3001 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
     3061 ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat
     3121 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
     3181 gggcactgac aattccgtgg tgttgtcggg gaagctgacg tcctttccat ggctgctcgc
     3241 ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa
     3301 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
     3361 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcctg gaaacggggg
     3421 aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata
     3481 aaaagacaga ataaaacgca cgggtgttgg gtcgtttgtt cataaacgcg gggttcggtc
     3541 ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt
     3601 ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa
     3661 cgtcggggcg gcaggccctg ccatagcaga tctgcgcagc tggggctcta gggggtatcc
     3721 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     3781 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     3841 cacgttcgcc ggctttcccc gtcaagctct aaatcggggc atccctttag ggttccgatt
     3901 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     3961 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     4021 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     4081 ataagggatt ttggggattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     4141 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     4201 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     4261 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     4321 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     4381 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
     4441 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     4501 ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc
     4561 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     4621 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     4681 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     4741 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     4801 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     4861 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     4921 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     4981 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     5041 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     5101 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     5161 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     5221 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     5281 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     5341 gacgagttct tctgagcggg actctggggt tcgcgaaatg accgaccaag cgacgcccaa
     5401 cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     5461 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     5521 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     5581 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     5641 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     5701 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     5761 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     5821 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     5881 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     5941 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     6001 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     6061 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     6121 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     6181 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     6241 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcaatg
     6301 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     6361 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     6421 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     6481 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     6541 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     6601 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     6661 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     6721 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     6781 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     6841 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     6901 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     6961 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     7021 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     7081 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     7141 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     7201 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     7261 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     7321 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     7381 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     7441 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     7501 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     7561 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     7621 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     7681 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     7741 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     7801 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga
     7861 cggatcggga gatctcccga tcccctatgg tcgactctca gtacaatctg ctctgatgcc
     7921 gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg
     7981 agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt
     8041 agg
//
