pIRESneo3 contains the internal ribosome entry site (IRES) of the encephalomyocarditis virus (ECMV), which permits the translation of two open reading frames from one messenger RNA (1–3). After selection with G418, nearly all surviving colonies will stably express the gene of interest, thus decreasing the need to screen large numbers of colonies to find functional clones. To select for cells that express high levels of the gene of interest, the selective pressure for antibiotic resistance was increased by shifting the neomycin phosphotransferase gene downstream to a less optimal position for translation as directed by the IRES sequence (1). By decreasing the level of expression of the antibiotic resistance marker, the selective pressure on the entire expression cassette is increased, resulting in the selection of cells that express the entire transcript, including the gene of interest, at high levels.
LOCUS Exported 5260 bp ds-DNA circular SYN 29-AUG-2016
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Untitled 10
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5260)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Monday, August 29, 2016 from SnapGene Viewer 3.1.4
FEATURES Location/Qualifiers
source 1..5260
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 235..614
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 615..818
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
promoter 863..881
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
intron 1051..1280
/note="chimeric intron"
/note="chimera between introns from adenovirus and
immunoglobulin heavy chain genes"
misc_feature 1338..1911
/note="IRES"
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
CDS 1947..2750
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/note="NeoR/KanR"
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
polyA_signal 2880..3001
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
primer_bind complement(3103..3119)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 3127..3143
/bound_moiety="lac repressor encoded by lacI"
/note="lac operator"
/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-beta-D-thiogalactopyranoside (IPTG)."
promoter complement(3151..3181)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 3196..3217
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(3505..4093)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(4264..5124)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(5125..5229)
/gene="bla"
/note="AmpR promoter"
ORIGIN
1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
901 gagctcggat cgatatctgc ggcctagcta gcgcttaagg cctgttaacc ggtcgtacgt
961 ctccggattc gaattcggat ccgcggccgc atagataact gatccagtgt gctggaatta
1021 attcgctgtc tgcgagggcc agctgttggg gtgagtactc cctctcaaaa gcgggcatga
1081 cttctgcgct aagattgtca gtttccaaaa acgaggagga tttgatattc acctggcccg
1141 cggtgatgcc tttgagggtg gccgcgtcca tctggtcaga aaagacaatc tttttgttgt
1201 caagcttgag gtgtggcagg cttgagatct ggccatacac ttgagtgaca atgacatcca
1261 ctttgccttt ctctccacag gtgtccactc ccaggtccaa ctgcaggtcg agcatgcatc
1321 tagggcggcc aattccgccc ctctccctcc ccccccccta acgttactgg ccgaagccgc
1381 ttggaataag gccggtgtgc gtttgtctat atgttatttt ccaccatatt gccgtctttt
1441 ggcaatgtga gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt
1501 tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg
1561 gaagcttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca
1621 cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg
1681 gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc
1741 tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct
1801 gatctggggc ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta
1861 ggccccccga accacgggga cgtggttttc ctttgaaaaa cacgatgata agcttgccac
1921 aacccgggat aattcctgca gccaatatgg gatcggccat tgaacaagat ggattgcacg
1981 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa
2041 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg
2101 tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt
2161 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa
2221 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc
2281 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg
2341 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg
2401 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg
2461 aactgttcgc caggctcaag gcgcgcatgc ccgacggcga tgatctcgtc gtgacccatg
2521 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact
2581 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg
2641 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc
2701 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga ggggatcaat
2761 tctctagata actgatcata atcagccata ccacatttgt agaggtttta cttgctttaa
2821 aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta
2881 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
2941 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
3001 aacgcgtcga gtgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
3061 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
3121 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
3181 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
3241 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
3301 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
3361 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
3421 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
3481 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
3541 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
3601 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
3661 gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct
3721 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
3781 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
3841 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
3901 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat
3961 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
4021 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
4081 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
4141 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
4201 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
4261 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
4321 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
4381 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
4441 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
4501 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
4561 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
4621 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
4681 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
4741 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
4801 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
4861 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
4921 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
4981 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
5041 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
5101 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
5161 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
5221 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
//
English