LOCUS       40924_30915        7868 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Reporter construct with TurboRFP and neomycin resistance markers, 
            for cloning 3' UTR sequences downstream of firefly luciferase to 
            validate miRNA targets.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7868)
  AUTHORS   OriGene
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 7868)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Compatible with the PrecisionShuttle(TM) system.
FEATURES             Location/Qualifiers
     source          1..7868
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        458..562
                     /label=AmpR promoter
     enhancer        564..755
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     rep_origin      751..886
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             935..1726
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGMADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     misc_feature    1750..2328
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     CDS             2334..3983
                     /codon_start=1
                     /label=luciferase
                     /note="firefly luciferase"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     polyA_signal    4285..4332
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4661..5249
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     polyA_signal    complement(5398..6020)
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     6031..6047
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(6067..6090)
                     /codon_start=1
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
                     /translation="DYKDDDDK"
     CDS             complement(6109..6138)
                     /codon_start=1
                     /label=Myc
                     /note="Myc (human c-Myc proto-oncogene) epitope tag"
                     /translation="EQKLISEEDL"
     CDS             complement(6150..6842)
                     /codon_start=1
                     /label=TurboRFP
                     /note="red fluorescent protein from Entacmaea quadricolor"
                     /translation="MSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMKIKV
                     VEGGPLPFAFDILATSFMYGSKAFINHTQGIPDFFKQSFPEGFTWERITTSEDGGVLTA
                     TQDTSFQNGCIIYNVKINGVNFPSNGPVMQKKTRGWEANTEMLYPADGGLRGHSQMALK
                     LVGGGYLHCSFKTTYRSKKPAKNLKMPGFHFVDHRLERIKEADKETYVEQHEMAVAKYC
                     DLPSKLGHR"
     promoter        complement(6899..6917)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(6943..7146)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     enhancer        complement(7147..7526)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(7687..7703)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      complement(join(7844..7868,1..431))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
       61 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
      121 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
      181 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
      241 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
      301 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
      361 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
      421 acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
      481 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
      541 ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag
      601 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
      661 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
      721 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc cagttccgcc
      781 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
      841 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
      901 gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
      961 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     1021 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     1081 caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg
     1141 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     1201 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     1261 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     1321 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     1381 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     1441 actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg
     1501 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     1561 tggccggctg ggtatggcgg atcgctatca ggacatagcg ttggctaccc gtgatattgc
     1621 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     1681 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
     1741 gggttcgatt ccgccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg
     1801 tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc
     1861 cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa
     1921 ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga
     1981 caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc
     2041 ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc
     2101 cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac
     2161 aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg
     2221 tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac
     2281 ggggacgtgg ttttcctttg aaaaacacga tgataatatg gccacaacaa ttgatggaag
     2341 acgccaaaaa cataaagaaa ggcccagcgc cattctaccc actcgaagac gggaccgccg
     2401 gcgagcagct gcacaaagcc atgaagcgct acgccctggt gcccggcacc atcgccttta
     2461 ccgacgcaca tatcgaggtg gacattacct acgccgagta cttcgagatg agcgttcggc
     2521 tggcagaagc tatgaagcgc tatgggctga atacaaacca tcggatcgtg gtgtgcagcg
     2581 agaatagctt gcagttcttc atgcccgtgt tgggtgccct gttcatcggt gtggctgtgg
     2641 ccccagctaa cgacatctac aacgagcgcg agctgctgaa cagcatgggc atcagccagc
     2701 ccaccgtcgt attcgtgagc aagaaagggc tgcaaaagat cctcaacgtg caaaagaagc
     2761 taccgatcat acaaaagatc atcatcatgg atagcaagac cgactaccag ggcttccaaa
     2821 gcatgtacac cttcgtgact tcccatttgc cacccggctt caacgagtac gacttcgtgc
     2881 ccgagagctt cgaccgggac aaaaccatcg ccctgatcat gaacagtagt ggcagtaccg
     2941 gattgcccaa gggcgtagcc ctaccgcacc gcaccgcttg tgtccgattc agtcatgccc
     3001 gcgaccccat cttcggcaac cagatcatcc ccgacaccgc tatcctcagc gtggtgccat
     3061 ttcaccacgg cttcggcatg ttcaccacgc tgggctactt gatctgcggc tttcgggtcg
     3121 tgctcatgta ccgcttcgag gaggagctat tcttgcgcag cttgcaagac tataagattc
     3181 aatctgccct gctggtgccc acactattta gcttcttcgc taagagcact ctcatcgaca
     3241 agtacgacct aagcaacttg cacgagatcg ccagcggcgg ggcgccgctc agcaaggagg
     3301 taggtgaggc cgtggccaaa cgcttccacc taccaggcat ccgccagggc tacggcctga
     3361 cagaaacaac cagcgccatt ctgatcaccc ccgaagggga cgacaagcct ggcgcagtag
     3421 gcaaggtggt gcccttcttc gaggctaagg tggtggactt ggacaccggt aagacactgg
     3481 gtgtgaacca gcgcggcgag ctgtgcgtcc gtggccccat gatcatgagc ggctacgtta
     3541 acaaccccga ggctacaaac gctctcatcg acaaggacgg ctggctgcac agcggcgaca
     3601 tcgcctactg ggacgaggac gagcacttct tcatcgtgga ccggctgaag agcctgatca
     3661 aatacaaggg ctaccaggta gccccagccg aactggagag catcctgctg caacacccca
     3721 acatcttcga cgccggggtc gccggcctgc ccgacgacga tgccggcgag ctgcccgccg
     3781 cagtcgtcgt gctggaacac ggtaaaacca tgaccgagaa ggagatcgtg gactatgtgg
     3841 ccagccaggt tacaaccgcc aagaagctgc gcggtggtgt tgtgttcgtg gacgaggtgc
     3901 ctaaaggact gaccggcaag ttggacgccc gcaagatccg cgagattctc attaaggcca
     3961 agaagggcgg aaagatcgcc gtgtaacaat tggcagagct cagaattcaa gcgatcgctt
     4021 ggcgcgcccg gaccgttacg cgtaagcggc cgcggcatct agattcgaag aaaatgaccg
     4081 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa
     4141 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     4201 tcatgctgga gttcttcgcc caccctaggg ggaggctaac tgaaacacgg aaggagacaa
     4261 taccggaagg aacccgcgct atgacggcaa taaaaagaca gaataaaacg cacggtgttg
     4321 ggtcgtttgt tcataaacgc ggggttcggt cccagggctg gcactctgtc gataccccac
     4381 cgagacccca ttggggccaa tacgcccgcg tttcttcctt ttccccaccc caccccccaa
     4441 gttcgggtga aggcccaggg ctcgcagcca acgtcggggc ggcaggccct gccatagcct
     4501 caggttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     4561 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     4621 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     4681 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     4741 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     4801 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     4861 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     4921 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     4981 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     5041 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     5101 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     5161 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     5221 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     5281 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     5341 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgctggccg cgtacccaat
     5401 tcaacaggca tctactgagt ggacccaacg catgagagga cagtgccaag caagcaactc
     5461 aaatgtccca ccggttgggc atggccaggt agcctatgct gtgtctggac gtcctcctgc
     5521 tggtatagtt attttaaaat cagaaggaca gggaagggag cagtggttca cgcctgtaat
     5581 cccagcaatt tgggaggcca aggtgggtag atcacctgag attaggagtt ggagaccagc
     5641 ctggccaata tggtgaaacc ccgtctctac caaaaaaaca aaaattagct gagcctggtc
     5701 atgcatgcct ggaatcccaa caactcggga ggctgaggca ggagaatcgc ttgaacccag
     5761 gaggcggaga ttgcagtgag ccaagattgt gccactgcac tccagcttgg ttcccaatag
     5821 accccgcagg ccctacaggt tgtcttccca acttgcccct tgctccatac cacccccctc
     5881 caccccataa tattatagaa ggacacctag tcagacaaaa tgatgcaact taattttatt
     5941 aggacaaggc tggtgggcac tggagtggca acttccaggg ccaggagagg cactggggag
     6001 gggtcacagg gatgccaccc gggatctgtt caggaaacag ctatgaccgc ggccggccgt
     6061 ttaaacctta tcgtcgtcat ccttgtaatc caggatatca tttgctgcca gatcctcttc
     6121 tgagatgagt ttctgctcga gggacgcgat ctgtgcccca gtttgctagg gaggtcgcag
     6181 tacttggcca cagccatctc gtgctgctcg acgtaggtct ctttgtcggc ctccttgatt
     6241 ctttccagtc tgtggtccac gaagtggaag ccgggcatct tgaggttctt agcgggtttc
     6301 ttggatctgt atgtggtctt gaaggagcag tgcaggtagc ccccgcccac gagcttcagg
     6361 gccatctggc tgtggcctct caggccgccg tcagcggggt acagcatctc ggtgttggcc
     6421 tcccagccgc gtgttttctt ctgcatcaca gggccgttgg atgggaagtt caccccgttg
     6481 atcttgacgt tgtagatgat gcagccgttc tggaagctgg tgtcctgggt agcggtcagc
     6541 acgcccccgt cttcggatgt ggtgattctc tcccatgtga agccctcagg gaaggactgc
     6601 ttaaagaagt cggggatgcc ctgggtgtgg ttgatgaagg ctttgctgcc gtacatgaag
     6661 ctggtagcca ggatgtcgaa ggcgaagggg agagggccgc cctcgaccac cttgatcttc
     6721 atggtctggg tgccctcgta gggcttgcct tcgccctcgg atgtgcactt gaagtggtgg
     6781 ttgttcacgg tgccctccat gtacagcttc atgtgcatgt tctccttgat cagctcgctc
     6841 atggcgaacg cggcgcagat ctcctcggta ctggatccag tcgacgattc ccggccgccc
     6901 tatagtgagt cgtattacaa aattctgacg gttcactaaa cgagctctgc ttatatagac
     6961 ctcccaccgt acacgcctac cgcccatttg cgtcaacggg gcggggttat tacgacattt
     7021 tggaaagtcc cgttgatttt ggtgccaaaa caaactccca ttgacgtcaa tggggtggag
     7081 acttggaaat ccccgtgagt caaaccgcta tccacgccca ttggtgtact gccaaaaccg
     7141 catcaccatg gtaatagcga tgactaatac gtagatgtac tgccaagtag gaaagtcccg
     7201 taaggtcatg tactgggcat aatgccaggc gggccattta ccgtcattga cgtcaatagg
     7261 gggcggactt ggcatatgat acacttgatg tactgccaag tgggcagttt accgtaaata
     7321 ctccacccat tgacgtcaat ggaaagtccc tattggcgtt actatgggaa catacgtcat
     7381 tattgacgtc aatgggcggg ggtcgttggg cggtcagcca ggcgggccat ttaccgtaag
     7441 ttatgtaacg cggaactcca tatatgggct atgaactaat gaccccgtaa ttgattacta
     7501 ttaataacta gtcaataatc aatgtcaaca tggcggtcat attggacatg agccaatata
     7561 aatgtacata ttatgatata gatacaacgt atgcaatggc caatagccaa tattgattta
     7621 tgctatataa ccaatgacta atatggctaa ttgccaatat tgattcaatg tatagatcag
     7681 cttggcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     7741 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     7801 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggaaattgt aagcgttaat
     7861 attttgtt
//
