LOCUS       V045854                 8403 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V045854
VERSION     V045854
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             744..1451
                     /codon_start=1
                     /note="mammalian codon-optimized"
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /transl_table=1
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
                     /label="mCherry"
     CDS             1500..3083
                     /label="Runx2(NM_001146038)"
                     /note="Runx2(NM_001146038)"
                     /gene="Runx2"
     CDS             3090..3155
                     /codon_start=1
                     /product="three tandem FLAG® epitope tags, followed by an
                     enterokinase cleavage site"
                     /transl_table=1
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
                     /label="3xFLAG"
     misc_feature    3179..3767
                     /note="woodchuck hepatitis virus&nbsp;posttranscriptional
                     regulatory element"
                     /label="WPRE"
     polyA_signal    3833..3881
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal&nbsp;(Cole and Stacy, 1985)"
                     /label="HSV TK poly(A) signal"
     rep_origin      4083..4511
                     /direction=RIGHT
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     promoter        4525..4854
                     /note="SV40 enhancer and early promoter"
                     /label="SV40 promoter"
     CDS             4921..5715
                     /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    5891..6012
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     primer_bind     complement(6061..6077)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    6085..6101
                     /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(6109..6139)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    6154..6175
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(6463..7051)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(7222..8082)
                     /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(8083..8187)
                     /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 tggtaccgcc accatggtga gcaagggcga ggaggataac atggccatca
      781 tcaaggagtt catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg
      841 agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg
      901 tgaccaaggg tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg
      961 gctccaaggc ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc
     1021 ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga
     1081 cccaggactc ctccctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca
     1141 acttcccctc cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctcctccg
     1201 agcggatgta ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga
     1261 aggacggcgg ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc
     1321 agctgcccgg cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact
     1381 acaccatcgt ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg
     1441 agctgtacaa gtggatcgga ggagggggaa gcggtggagg cggtagtgag ctcggatcca
     1501 tggcgtcaaa cagcctcttc agcgcagtga caccgtgtca gcaaagcttc ttttgggatc
     1561 cgagcaccag ccggcgcttc agccccccct ccagcagcct gcagcccggc aagatgagcg
     1621 acgtgagccc ggtggtggct gcgcagcagc agcaacagca gcagcagcag cagcaacagc
     1681 agcagcaaca acagcaacag caacaacagc agcagcagca gcagcagcag gaggcggccg
     1741 cagcagcagc ggcggcagcg gcggcggcag cagcggcggc ggccgcagtg ccccgattga
     1801 ggccgccgca cgacaaccgc accatggtgg agatcatcgc ggaccacccg gccgaactgg
     1861 tccgcaccga cagtcccaac ttcctgtgct ccgtgctgcc ctcgcactgg cggtgcaaca
     1921 agaccctgcc cgtggccttc aaggttgtag ccctcggaga ggtaccagat gggactgtgg
     1981 ttaccgtcat ggccgggaat gatgagaact actccgccga gctccgaaat gcctccgctg
     2041 ttatgaaaaa ccaagtagcc aggttcaacg atctgagatt tgtgggccgg agcggacgag
     2101 gcaagagttt caccttgacc ataacagtct tcacaaatcc tccccaagtg gccacttacc
     2161 acagagctat taaagtgaca gtggacggtc cccgggaacc aagaaggcac agacagaagc
     2221 ttgatgactc taaacctagt ttgttctctg atcgcctcag tgatttaggg cgcattcctc
     2281 atcccagtat gagagtaggt gtcccgcctc agaacccacg gccctccctg aactctgcac
     2341 caagtccttt taatccacaa ggacagagtc agattacaga tcccaggcag gcacagtctt
     2401 ccccaccgtg gtcctatgac cagtcttacc cctcctatct gagccagatg acatccccat
     2461 ccatccactc caccacgccg ctgtcttcca cacggggcac cgggctacct gccatcactg
     2521 acgtgcccag gcgtatttca gatgatgaca ctgccacctc tgacttctgc ctctggcctt
     2581 cctctctcag taagaagagc caggcaggtg cttcagaact gggccctttt tcagacccca
     2641 ggcagttccc aagcatttca tccctcactg agagccgctt ctccaaccca cgaatgcact
     2701 acccagccac ctttacctac accccgccag tcacgtcagg catgtccctc ggcatgtccg
     2761 ccaccactca ctaccacacg tacctgccac caccctaccc cggctcttcc caaagccaga
     2821 gtggaccctt ccagaccagc agcactccat atctctacta tggtacttcg tcagcatcct
     2881 atcagttccc aatggtaccc gggggagacc ggtctccttc caggatggtc ccaccatgca
     2941 ccaccacctc gaatggcagc acgctattaa atccaaattt gcctaaccag aatgatggtg
     3001 ttgacgctga cggaagccac agcagttccc caactgtttt gaattctagc ggcagaatgg
     3061 atgagtctgt ttggcggcca tatctcgagg actacaaaga ccatgacggt gattataaag
     3121 atcatgacat cgactacaag gatgacgatg acaagtagtg agggcccgat atctcgacaa
     3181 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
     3241 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
     3301 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg
     3361 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
     3421 ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat
     3481 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
     3541 gggcactgac aattccgtgg tgttgtcggg gaagctgacg tcctttccat ggctgctcgc
     3601 ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa
     3661 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
     3721 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcctg gaaacggggg
     3781 aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata
     3841 aaaagacaga ataaaacgca cgggtgttgg gtcgtttgtt cataaacgcg gggttcggtc
     3901 ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt
     3961 ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa
     4021 cgtcggggcg gcaggccctg ccatagcaga tctgcgcagc tggggctcta gggggtatcc
     4081 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     4141 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     4201 cacgttcgcc ggctttcccc gtcaagctct aaatcggggc atccctttag ggttccgatt
     4261 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     4321 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     4381 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     4441 ataagggatt ttggggattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     4501 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     4561 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     4621 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     4681 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     4741 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
     4801 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     4861 ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc
     4921 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     4981 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     5041 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     5101 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     5161 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     5221 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     5281 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     5341 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     5401 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     5461 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     5521 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     5581 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     5641 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     5701 gacgagttct tctgagcggg actctggggt tcgcgaaatg accgaccaag cgacgcccaa
     5761 cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     5821 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     5881 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     5941 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     6001 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     6061 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     6121 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     6181 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     6241 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     6301 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     6361 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     6421 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     6481 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     6541 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     6601 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcaatg
     6661 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     6721 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     6781 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     6841 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     6901 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     6961 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     7021 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     7081 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     7141 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     7201 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     7261 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     7321 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     7381 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     7441 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     7501 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     7561 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     7621 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     7681 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     7741 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     7801 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     7861 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     7921 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     7981 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     8041 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     8101 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     8161 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga
     8221 cggatcggga gatctcccga tcccctatgg tcgactctca gtacaatctg ctctgatgcc
     8281 gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg
     8341 agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt
     8401 agg
//
