LOCUS       V032970                 7959 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V032970
VERSION     V032970
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..2639
                     /label="KLHL14(NM_020805)"
                     /note="KLHL14(NM_020805)"
                     /gene="KLHL14"
     CDS             2646..2711
                     /codon_start=1
                     /product="three tandem FLAG® epitope tags, followed by an
                     enterokinase cleavage site"
                     /transl_table=1
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
                     /label="3xFLAG"
     misc_feature    2735..3323
                     /note="woodchuck hepatitis virus&nbsp;posttranscriptional
                     regulatory element"
                     /label="WPRE"
     polyA_signal    3389..3437
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal&nbsp;(Cole and Stacy, 1985)"
                     /label="HSV TK poly(A) signal"
     rep_origin      3639..4067
                     /direction=RIGHT
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     promoter        4081..4410
                     /note="SV40 enhancer and early promoter"
                     /label="SV40 promoter"
     CDS             4477..5271
                     /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    5447..5568
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     primer_bind     complement(5617..5633)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    5641..5657
                     /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(5665..5695)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    5710..5731
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(6019..6607)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(6778..7638)
                     /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(7639..7743)
                     /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 ccaccatgtc cagatccggg gacaggacct
      781 ccaccttcga ccccagccac agcgacaacc tgctgcacgg cctcaacctg ctgtggagga
      841 agcagctgtt ttgcgacgtg accctgacgg cccagggcca gcagttccat tgccacaagg
      901 ccgtgctggc ctcctgctcg cagtacttcc gatcgctctt ctccagccac ccccctctcg
      961 ggggaggggt cggcggccag gacggcctgg gggcccccaa ggaccagcag cagccgccgc
     1021 agcagcagcc gtcacagcag cagcagccgc cgccgcagga ggagcccggg actccttctt
     1081 cctcccccga cgacaagctg ctgaccagcc cccgggccat caacaacctg gtgctgcagg
     1141 gctgctcgtc catcgggctg cgcctggtgc tcgagtacct ctacacggcc aacgtgaccc
     1201 tgtccctgga cacggtggag gaggtgctgt cggtcagcaa gatcctgcac atcccccagg
     1261 tcaccaagct ctgcgtgcag ttcctcaacg accagatctc ggtgcagaac tacaagcagg
     1321 tgtgcaagat cgccgcgctg cacggcctgg aggagaccaa gaagctggcc aacaagtacc
     1381 tggtggagga tgtgctgctg ctcaacttcg aggagatgcg cgccctgctg gactcgctgc
     1441 cgccccccgt ggagtcggag ctggcgctct tccagatgtc cgtgctgtgg ctggagcacg
     1501 accgcgagac ccgcatgcag tatgcgcctg acctcatgaa gcgcctccgc ttcgccctca
     1561 tcccggcccc ggagctggtg gagcgggtcc agtcagtgga tttcatgcga accgacccgg
     1621 tctgccagaa gctgctgctg gacgccatga actaccacct gatgcccttc aggcagcact
     1681 gcaggcagag cctggccagc agaattcgct ctaacaagaa aatgctgtta ttggttggag
     1741 ggctgcctcc tggaccggac cggctcccca gcaatttggt tcagtattac gacgatgaaa
     1801 agaagacatg gaaaatactc acaattatgc catacaacag tgcccaccac tgcgttgtgg
     1861 aggtggaaaa cttcttgttc gtgttgggtg gagaggacca gtggaatccg aatggaaaac
     1921 acagtacaaa ttttgtcagc cgatatgatc ctcgatttaa tagctggatt caacttccac
     1981 ccatgcagga aagaagagcc agtttctatg catgtcggtt ggacaagcat ttatacgtaa
     2041 ttggtggaag gaatgaaact ggctacttgt ccagcgtgga gtgctataac ctagaaacga
     2101 atgaatggcg ctatgtgtcc tctttgccac agcctctggc ggctcatgcg ggagcagtgc
     2161 acaatgggaa aatatacatt tcagggggtg tacacaatgg agaatatgtc ccatggctat
     2221 attgctatga cccagtaatg gatgtctggg ctcgaaaaca agatatgaac acaaaacgtg
     2281 caattcacac tttggctgta atgaatgatc gcttgtatgc aattggagga aatcatttga
     2341 aaggtttctc ccaccttgat gtaatgcttg tggaatgcta tgacccaaaa ggtgaccagt
     2401 ggaatatact ccaaactccc attttggagg gtcgaagtgg ccctggctgt gcagtgcttg
     2461 atgacagcat ttaccttgtg ggaggctaca gctggagtat gggggcctac aagtcatcta
     2521 caatatgcta ttgtccagag aaaggaacct ggacagaact cgaaggagat gtagcagaac
     2581 cgttggcagg ccctgcctgt gtgacagtta ttctgccctc ttgtgtacca tacaacaaag
     2641 gatccgacta caaagaccat gacggtgatt ataaagatca tgacatcgac tacaaggatg
     2701 acgatgacaa gtagtgaggg cccgatatct cgacaatcaa cctctggatt acaaaatttg
     2761 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc
     2821 tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta
     2881 taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt
     2941 ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca
     3001 gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc
     3061 ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt
     3121 gtcggggaag ctgacgtcct ttccatggct gctcgcctgt gttgccacct ggattctgcg
     3181 cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg
     3241 cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat
     3301 ctccctttgg gccgcctccc cgcctggaaa cgggggaggc taactgaaac acggaaggag
     3361 acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggg
     3421 tgttgggtcg tttgttcata aacgcggggt tcggtcccag ggctggcact ctgtcgatac
     3481 cccaccgaga ccccattggg gccaatacgc ccgcgtttct tccttttccc caccccaccc
     3541 cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag gccctgccat
     3601 agcagatctg cgcagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     3661 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     3721 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     3781 agctctaaat cggggcatcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     3841 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     3901 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     3961 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgg ggatttcggc
     4021 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     4081 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     4141 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     4201 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     4261 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     4321 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     4381 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     4441 ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     4501 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     4561 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     4621 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     4681 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     4741 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     4801 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     4861 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     4921 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     4981 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
     5041 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     5101 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     5161 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     5221 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     5281 tggggttcgc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     5341 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     5401 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
     5461 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     5521 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat
     5581 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     5641 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     5701 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     5761 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     5821 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     5881 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     5941 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     6001 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     6061 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     6121 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     6181 ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc
     6241 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     6301 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     6361 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     6421 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc
     6481 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     6541 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     6601 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     6661 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     6721 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     6781 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     6841 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     6901 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     6961 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     7021 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     7081 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     7141 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     7201 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     7261 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     7321 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     7381 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     7441 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     7501 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     7561 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     7621 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     7681 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     7741 gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc
     7801 ctatggtcga ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc
     7861 cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg
     7921 caaggcttga ccgacaattg catgaagaat ctgcttagg
//
