LOCUS       V042165                 7923 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V042165
VERSION     V042165
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..2603
                     /label="Sphk2(NM_001012066)"
                     /note="Sphk2(NM_001012066)"
                     /gene="Sphk2"
     CDS             2610..2675
                     /codon_start=1
                     /product="three tandem FLAG® epitope tags, followed by an
                     enterokinase cleavage site"
                     /transl_table=1
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
                     /label="3xFLAG"
     misc_feature    2699..3287
                     /note="woodchuck hepatitis virus&nbsp;posttranscriptional
                     regulatory element"
                     /label="WPRE"
     polyA_signal    3353..3401
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal&nbsp;(Cole and Stacy, 1985)"
                     /label="HSV TK poly(A) signal"
     rep_origin      3603..4031
                     /direction=RIGHT
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     promoter        4045..4374
                     /note="SV40 enhancer and early promoter"
                     /label="SV40 promoter"
     CDS             4441..5235
                     /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    5411..5532
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     primer_bind     complement(5581..5597)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    5605..5621
                     /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(5629..5659)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    5674..5695
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(5983..6571)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(6742..7602)
                     /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(7603..7707)
                     /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 ccaccatggc cccaccacca ctactgccag
      781 tggctgccag cactccgatc ctgcacggcg agtttggttc ctacccagcc catggcccca
      841 ggtttgccct caccctcacg acacaagccc tacacataca gcgactgcgc ccaaagccag
      901 aagcccggcc ccgagatggt ctagtccctc tggacgaggt ctcaggctgt ggcactctgc
      961 agagccgtag ccccgaagac actgcagcct acttctgcat ctacacctac ccacgtggcc
     1021 ggcgtggggg ccggcgcaga gctactcgta ccttccgggc cgacggggcc gccacttatg
     1081 tggagaatcg tgcagaggcc cagcgttggg caactgctct catgtgtctc ctccgaggag
     1141 tgcctctgtc tggggaccag gaaatcaccc ctgaattgct gccccggaag cccaggctgc
     1201 tcctattggt caatcctttt ggggggcggg gcctggcctg gcagcgctgt atggaccacg
     1261 tggtgcccat gatctctgaa gctgggctgt ccttcaacct catacagaca gaacgacaaa
     1321 accatgcacg tgagctggtg caggggttga gcctgagtga gtgggaaggc attgtcactg
     1381 tgtccggaga tgggctgctt tatgaggtgc tgaatgggct cctcgaccgc ccagattggg
     1441 aagacgctgt gcggatgccc attggtgtcc tcccctgtgg ttcgggcaat gcactagctg
     1501 gggcagttaa ccatcatggc gggtttgagc aaactgttgg tgttgacctg ttgctcaact
     1561 gctcgcttct tctctgccgg ggtggcagcc atcctctgga cctgctctct gtgacgctag
     1621 cctcaggatc ccgctgtttt tccttcctgt cagttgcctg gggattcttg tcagatgtgg
     1681 acattcacag cgagcgcttc agagccctgg gcagtgctcg attcacactg ggtgcagtgc
     1741 taggcctggc cacattgcat acctaccgtg gacgtctctc ctacctccct gctaccacag
     1801 aaccagcctt gcccatccca ggccatggtc tgccaagagc caagtcagaa ctagccttgg
     1861 ccccagcccc agccccagcc gccacccact cacctcttca tcgatctgtg tctgacctgc
     1921 ccctgcctct tccccagcct gccctggcct cccctggctc ccctgagccc ctgcctgacc
     1981 tgtccctcaa tggtggtggt ccagagctga ctggagactg ggggggagct ggggatgcac
     2041 ctttgtcccc agacccattg ctgccttcat cccccaacgc tttcaaaaca gctcagcttt
     2101 cacccatcgc cgaagggccc tcagaaatgc cagcatcagg gctgctgcct cccatccaca
     2161 atgctccaga agcctctacc tggggcccag cggaccacct gctccctccc ctgggctctc
     2221 cactgcccca agactgggtg acaatagagg gggagtttgt actcatgttg gccatcttgc
     2281 caagccacct ctgcgcagac ctgatggcag ccccacacgc gcgttttgac gatggtgtgg
     2341 tgcacctgtg ctgggtgcgg agcggcatct cacgggccgc acttctacga attttgctgg
     2401 ccatggaaca tggaaaccac ttcagcctgg gctgcccaca tctgggctat gctgcagcac
     2461 gtgccttccg ccttgagcca ctcacgcctc gtggggtgct cactgtagat ggggaattag
     2521 tagagtatgg gccaatacag gcacaggtgc atccaagtct cgccacactg ctcactgggc
     2581 ctgcaggtca aaagcctcga gccggatccg actacaaaga ccatgacggt gattataaag
     2641 atcatgacat cgactacaag gatgacgatg acaagtagtg agggcccgat atctcgacaa
     2701 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
     2761 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
     2821 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg
     2881 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
     2941 ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat
     3001 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
     3061 gggcactgac aattccgtgg tgttgtcggg gaagctgacg tcctttccat ggctgctcgc
     3121 ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa
     3181 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
     3241 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcctg gaaacggggg
     3301 aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata
     3361 aaaagacaga ataaaacgca cgggtgttgg gtcgtttgtt cataaacgcg gggttcggtc
     3421 ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt
     3481 ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa
     3541 cgtcggggcg gcaggccctg ccatagcaga tctgcgcagc tggggctcta gggggtatcc
     3601 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     3661 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     3721 cacgttcgcc ggctttcccc gtcaagctct aaatcggggc atccctttag ggttccgatt
     3781 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     3841 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     3901 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     3961 ataagggatt ttggggattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     4021 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     4081 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     4141 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     4201 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     4261 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
     4321 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     4381 ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc
     4441 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     4501 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     4561 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     4621 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     4681 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     4741 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     4801 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     4861 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     4921 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     4981 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     5041 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     5101 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     5161 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     5221 gacgagttct tctgagcggg actctggggt tcgcgaaatg accgaccaag cgacgcccaa
     5281 cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     5341 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     5401 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     5461 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     5521 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     5581 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     5641 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     5701 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     5761 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     5821 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     5881 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     5941 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     6001 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     6061 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     6121 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcaatg
     6181 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     6241 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     6301 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     6361 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     6421 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     6481 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     6541 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     6601 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     6661 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     6721 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     6781 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     6841 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     6901 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     6961 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     7021 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     7081 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     7141 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     7201 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     7261 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     7321 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     7381 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     7441 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     7501 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     7561 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     7621 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     7681 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga
     7741 cggatcggga gatctcccga tcccctatgg tcgactctca gtacaatctg ctctgatgcc
     7801 gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg
     7861 agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt
     7921 agg
//
