pRF-HUE

01/08/10

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Restriction map of pRF-HUE

pRF-HUE vector sequence in GenBank format

 

LOCUS pRF-HUE. 8709 bp DNA circular 4-JUL-2008
DEFINITION Complementary copy of pRF-HUE.
SOURCE
ORGANISM
COMMENT The vector can be used for ectopic over expression of genes in filamentous fungi.

The vector has been designed for the directional cloning of a single PCR
amplicon (typically a CDS plus terminator minus the start codon) after the
strong constitutive GAPDH promoter form Aspergillus nidulans, utilizing the
USER friendly cloning technology from NEB.

Add the following 5' overhangs to the primers for amplification of the two gene
specific homologous recombination flanks:

Gene-U1 (LB forward) 5- GGGTTTAAU - Forward primer sequence
Gene-U2 (LB reverse) 5'- GGACTTAAUG - Reverse primer sequence

The U2 primer contains a AUG (= ATG) which should be use as a start codon
in the gene you are trying to over express. Note that the vector does not contain
a 3'UTR or terminator, why these should be included in the PCR amplicon.

Digestion of vector prior to cloning:
1. Digest over night with PacI
2. Digest for 2 hours with Nt.Bbv.C IB (nicking enzyme)

Transformation of the vector into the fungi:
The vector is originate designed for Agrobacterium tumefaciens mediated
transformation, but can also be used for protoplast transformation (bipartial
transformation strategies).

COMMENT This file is created by Vector NTI
http://www.invitrogen.com/
COMMENT ORIGDB|EMBL
COMMENT VNTDATE|486999405|
COMMENT VNTDBDATE|487005772|
COMMENT LSOWNER|
COMMENT VNTNAME|pRF-HUE.|
COMMENT VNTAUTHORNAME|Rasmus J.N. Frandsen|
COMMENT VNTAUTHOREML|raf@kvl.dk|
COMMENT VNTREPLTYPE|Plasmid
COMMENT VNTEXTCHREPL|Bacteria
FEATURES Location/Qualifiers
primer_bind 8137..8162
/vntifkey="28"
/label=RF-3
primer_bind complement(3662..3685)
/vntifkey="28"
/label=RF-2
primer_bind 5892..5915
/vntifkey="28"
/label=RF-1
rep_origin complement(2657..3274)
/vntifkey="33"
/label=oriV
misc_feature complement(8391..8416)
/vntifkey="21"
/label=UCS\(LB)
promoter 6086..8387
/vntifkey="29"
/label=PgpdA
misc_structure complement(8688..8709)
/vntifkey="88"
/label=LB
CDS complement(127..1276)
/vntifkey="4"
/label=TrfA
CDS complement(1575..2370)
/vntifkey="4"
/label=KanR
CDS complement(4592..5637)
/vntifkey="4"
/label=hph
terminator complement(3872..4588)
/vntifkey="43"
/label=TtrpC
misc_structure complement(3422..3444)
/vntifkey="88"
/label=RB
promoter complement(5638..6002)
/vntifkey="29"
/label=pTrpC
misc_feature 3419..8682
/vntifkey="21"
/label=T-DNA
BASE COUNT 2007 a 2409 c 2199 g 2094 t
ORIGIN
1 caccagccag ccaacagctc cccgaccggc agctcggcac aaaatcacca ctcgatacag
61 gcagcccatc agtccgggac ggcgtcagcg ggagagcaga tccagccgga actgacccca
121 caaggcccta gcgtttgcaa tgcaccaggt catcattgac ccaggcgtgt tccaccaggc
181 cgctgcctcg caactcttcg caggcttcgc cgacctgctc gcgccacttc ttcacgcggg
241 tggaatccga tccgcacatg aggcggaagg tttccagctt gagcgggtac ggctcccggt
301 gcgagctgaa atagtcgaac atccgtcggg ccgtcggcga cagcttgcgg tacttctccc
361 atatgaattt cgtgtagtgg tcgccagcaa acagcacgac gatttcctcg tcgatcagga
421 cctggcaacg ggacgttttc ttgccacggt ccaggacgcg gaagcggtgc agcagcgaca
481 ccgattccag gtgcccaacg cggtcggacg tgaagcccat cgccgtcgcc tgtaggcgcg
541 acaggcattc ctcggccttc gtgtaatacc ggccattgat cgaccagccc aggtcctggc
601 aaagctcgta gaacgtgaag gtgatcggct cgccgatagg ggtgcgcttc gcgtactcca
661 acacctgctg ccacaccagt tcgtcatcgt cggcccgcag ctcgacgccg gtgtaggtga
721 tcttcacgtc cttgttgacg tggaaaatga ccttgttttg cagcgcctcg cgcgggattt
781 tcttgttgcg cgtggtgaac agggcagagc gggccgtgtc gtttggcatc gctcgcatcg
841 tgtccggcca cggcgcaata tcgaacaagg aaagctgcat ttccttgatc tgctgcttcg
901 tgtgtttcag caacgcggcc tgcttggcct cgctgacctg ttttgccagg tcctcgccgg
961 cggtttttcg cttcttggtc gtcatagttc ctcgcgtgtc gatggtcatc gacttcgcca
1021 aacctgccgc ctcctgttcg agacgacgcg aacgctccac ggcggccgat ggcgcgggca
1081 gggcaggggg agccagttgc acgctgtcgc gctcgatctt ggccgtagct tgctggacca
1141 tcgagccgac ggactggaag gtttcgcggg gcgcacgcat gacggtgcgg cttgcgatgg
1201 tttcggcatc ctcggcggaa aaccccgcgt cgatcagttc ttgcctgtat gccttccggt
1261 caaacgtccg attcattcac cctccttgcg ggattgcccc gactcacgcc ggggcaatgt
1321 gcccttattc ctgatttgac ccgcctggtg ccttggtgtc cagataatcc accttatcgg
1381 caatgaagtc ggtcccgtag accgtctggc cgtccttctc gtacttggta ttccgaatct
1441 tgccctgcac gaataccagc gaccccttgc ccaaatactt gccgtgggcc tcggcctgag
1501 agccaaaaca cttgatgcgg aagaagtcgg tgcgctcctg cttgtcgccg gcatcgttgc
1561 gccacatcta ggtactaaaa caattcatcc agtaaaatat aatattttat tttctcccaa
1621 tcaggcttga tccccagtaa gtcaaaaaat agctcgacat actgttcttc cccgatatcc
1681 tccctgatcg accggacgca gaaggcaatg tcataccact tgtccgccct gccgcttctc
1741 ccaagatcaa taaagccact tactttgcca tctttcacaa agatgttgct gtctcccagg
1801 tcgccgtggg aaaagacaag ttcctcttcg ggcttttccg tctttaaaaa atcatacagc
1861 tcgcgcggat ctttaaatgg agtgtcttct tcccagtttt cgcaatccac atcggccaga
1921 tcgttattca gtaagtaatc caattcggct aagcggctgt ctaagctatt cgtataggga
1981 caatccgata tgtcgatgga gtgaaagagc ctgatgcact ccgcatacag ctcgataatc
2041 ttttcagggc tttgttcatc ttcatactct tccgagcaaa ggacgccatc ggcctcactc
2101 atgagcagat tgctccagcc atcatgccgt tcaaagtgca ggacctttgg aacaggcagc
2161 tttccttcca gccatagcat catgtccttt tcccgttcca catcataggt ggtcccttta
2221 taccggctgt ccgtcatttt taaatatagg ttttcatttt ctcccaccag cttatatacc
2281 ttagcaggag acattccttc cgtatctttt acgcagcggt atttttcgat cagttttttc
2341 aattccggtg atattctcat tttagccatt tattatttcc ttcctctttt ctacagtatt
2401 taaagatacc ccaagaagct aattataaca agacgaactc caattcactg ttccttgcat
2461 tctaaaacct taaataccag aaaacagctt tttcaaagtt gttttcaaag ttggcgtata
2521 acatagtatc gacggagccg attttgaaac cacaattatg ggtgatgctg ccaacttact
2581 gatttagtgt atgatggtgt ttttgaggtg ctccagtggc gtacgcctgg ggggatggga
2641 ggcccgcgtt agcgggccgg gagggttcga gaaggggggg cacccccctt cggcgtgcgc
2701 ggtcacgcgc acagggcgca gccctggtta aaaacaaggt ttataaatat tggtttaaaa
2761 gcaggttaaa agacaggtta gcggtggccg aaaaacgggc ggaaaccctt gcaaatgctg
2821 gattttctgc ctgtggacag cccctcaaat gtcaataggt gcgcccctca tctgtcagca
2881 ctctgcccct caagtgtcaa ggatcgcgcc cctcatctgt cagtagtcgc gcccctcaag
2941 tgtcaatacc gcagggcact tatccccagg cttgtccaca tcatctgtgg gaaactcgcg
3001 taaaatcagg cgttttcgcc gatttgcgag gctggccagc tccacgtcgc cggccgaaat
3061 cgagcctgcc cctcatctgt caacgccgcg ccgggtgagt cggcccctca agtgtcaacg
3121 tccgcccctc atctgtcagt gagggccaag ttttccgcga ggtatccaca acgccggcgg
3181 ccgcggtgtc tcgcacacgg cttcgacggc gtttctggcg cgtttgcagg gccatagacg
3241 gccgccagcc cagcggcgag ggcaaccagc ccggtgagcg tcgcaaaggc gctcggtctt
3301 gccttgctcg tcggatctgg aaccctgtgg ttggcatgaa caaacaaatg gacgaacgga
3361 taaacctttt cacgcccttt taaatatccg attattctaa taaacgctct tttctcttag
3421 gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac
3481 aatctgatca tgagcggaga attaagggag tcacgaagct aaatccgttt aaacggcgcg
3541 ccggccggcc gcgatcgcgc actagtcggg ggatcctcta gatctcgacg ctctccctta
3601 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
3661 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
3721 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
3781 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc
3841 acgatgcgtc cggcgtagag gatcctctag aaagaaggat tacctctaaa caagtgtacc
3901 tgtgcattct gggtaaacga ctcataggag agttgtaaaa aagtttcggc cggcgtattg
3961 ggtgttacgg agcattcact aggcaaccat ggttactatt gtatacccat cttagtagga
4021 atgattttcg aggtttatac ctacgatgaa tgtgtgtcct gtaggcttga gagttcaagg
4081 aagaaacagt gcaattatct ttgcgaaccc aggggctggt gacggaattt tcatagtcaa
4141 gctatcagag taaagaagag gagcatgtca aagtacaatt agagacaaat atatagtcgc
4201 gtggagccaa gagcggattc ctcagtctcg taggtctctt gacgaccgtt gatctgcttg
4261 atctcgtctc ccgaaaatga aaatagactc tgctaagcta ttcttctgct tcgccggagc
4321 ctgaagggcg tactagggtt gcgaggtcca atgcattaat gcattgcaga tgagctgtat
4381 ctggaagagg taaacccgaa acgcgtttta ttcttgttga catggagcta ttaaatcact
4441 agaaggcact ctttgctgct tggacaaatg aacgtatctt atcgagatcc tgaacaccat
4501 ttgtctcaac tccggagctg acatcgacac caacgatctt atatccagat tcgtcaagct
4561 gtttgatgat ttcagtaacg ttaagtggat cccggtcggc atctactcta ttcctttgcc
4621 ctcggacgag tgctggggcg tcggtttcca ctatcggcga gtacttctac acagccatcg
4681 gtccagacgg ccgcgcttct gcgggcgatt tgtgtacgcc cgacagtccc ggctccggat
4741 cggacgattg cgtcgcatcg accctgcgcc caagctgcat catcgaaatt gccgtcaacc
4801 aagctctgat agagttggtc aagaccaatg cggagcatat acgcccggag ccgcggcgat
4861 cctgcaagct ccggatgcct ccgctcgaag tagcgcgtct gctgctccat acaagccaac
4921 cacggcctcc agaagaagat gttggcgacc tcgtattggg aatccccgaa catcgcctcg
4981 ctccagtcaa tgaccgctgt tatgcggcca ttgtccgtca ggacattgtt ggagccgaaa
5041 tccgcgtgca cgaggtgccg gacttcgggg cagtcctcgg cccaaagcat cagctcatcg
5101 agagcctgcg cgacggacgc actgacggtg tcgtccatca cagtttgcca gtgatacaca
5161 tggggatcag caatcgcgca tatgaaatca cgccatgtag tgtattgacc gattccttgc
5221 ggtccgaatg ggccgaaccc gctcgtctgg ctaagatcgg ccgcagcgat cgcatccatg
5281 gcctccgcga ccggctgcag aacagcgggc agttcggttt caggcaggtc ttgcaacgtg
5341 acaccctgtg cacggcggga gatgcaatag gtcaggctct cgctgaattc cccaatgtca
5401 agcacttccg gaatcgggag cgcggccgat gcaaagtgcc gataaacata acgatctttg
5461 tagaaaccat cggcgcagct atttacccgc aggacatatc cacgccctcc tacatcgaag
5521 ctgaaagcac gagattcttc gccctccgag agctgcatca ggtcggagac gctgtcgaac
5581 ttttcgatca gaaacttctc gacagacgtc gcggtgagtt caggcttttt catatcgatg
5641 cttgggtaga ataggtaagt cagattgaat ctgaaataaa gggaggaagg gcgaacttaa
5701 gaaggtatga ccgggtcgtt cacttacctt gcttgacaaa cgcaccaagt tatcgtgcac
5761 caagcagcag atgataataa tgtcctcgtt cctgtctgct aataagagtc acacttcgag
5821 cgccgccgct actgctacaa gtggggctga tctgaccagt tgcctaaatg aaccatcttg
5881 tcaaacgaca caaattttgt gctcaccgcc tggacgacta aaccaaaata ggcattcatt
5941 gttgacctcc actagctcca gccaagccca aaaagtgctc cttcaatatc atcttctgtc
6001 gaggcctgat catcgatggg cccatcgatg atcaggcctc gagatctaga ggatccttgc
6061 gcgcctaggc ggccgtggcc agcccgaatt cccttgtatc tctacacaca ggctcaaatc
6121 aataagaaga acggttcgtc tttttcgttt atatcttgca tcgtcccaaa gctattggcg
6181 ggatattctg tttgcagttg gctgacttga agtaatctct gcagatcttt cgacactgaa
6241 atacgtcgag cctgctccgc ttggaagcgg cgaggagcct cgtcctgtca caactaccaa
6301 catggagtac gataagggcc agttccgcca gctcattaag agccagttca tgggcgttgg
6361 catgatggcc gtcatgcatc tgtacttcaa gtacaccaac gctcttctga tccagtcgat
6421 catccgctga aggcgctttc gaatctggtt aagatccacg tcttcgggaa gccagcgact
6481 ggtgacctcc agcgtccctt taaggctgcc aacagctttc tcagccaggg ccagcccaag
6541 accgacaagg cctccctcca gaacgccgag aagaactgga ggggtggtgt caaggaggag
6601 taagctcctt attgaagtcg gaggacggag cggtgtcaag aggatattct tcgactctgt
6661 attatagata agatgatgag gaattggagg tagcatagct tcatttggat ttgctttcca
6721 ggctgagact ctagcttgga gcatagaggg tcctttggct ttcaatattc tcaagtatct
6781 cgagtttgaa cttattccct gtgaaccttt tattcaccaa tgagcattgg aatgaacatg
6841 aatctgagga ctgcaatcgc catgaggttt tcgaaataca tccggatgtc gaaggcttgg
6901 ggcacctgcg ttggttgaat ttagaacgtg gcactattga tcatccgata gctctgcaaa
6961 gggcgttgca caatgcaagt caaacgttgc tagcagttcc aggtggaatg ttatgatgag
7021 cattgtatta aatcaggaga tatagcatga tctctagtta gctcaccaca aaagtcagac
7081 ggcgtaacca aaagtcacac aacacaagct gtaaggattt cggcacggct acggaagacg
7141 gagaagccac cttcagtgga ctcgagtacc atttaattct atttgtgttt gatcgagacc
7201 taatacagcc cctacaacga ccatcaaagt cgtatagcta ccagtgagga agtggactca
7261 aatcgacttc agcaacatct cctggataaa ctttaagcct aaactataca gaataagata
7321 ggtggagagc ttataccgag ctcccaaatc tgtccagatc atggttgacc ggtgcctgga
7381 tcttcctata gaatcatcct tattcgttga cctagctgat tctggagtga cccagagggt
7441 catgacttga gcctaaaatc cgccgcctcc accatttgta gaaaaatgtg acgaactcgt
7501 gagctctgta cagtgaccgg tgactctttc tggcatgcgg agagacggac ggacgcagag
7561 agaagggctg agtaataagc cactggccag acagctctgg cggctctgag gtgcagtgga
7621 tgattattaa tccgggaccg gccgcccctc cgccccgaag tggaaaggct ggtgtgcccc
7681 tcgttgacca agaatctatt gcatcatcgg agaatatgga gcttcatcga atcaccggca
7741 gtaagcgaag gagaatgtga agccaggggt gtatagccgt cggcgaaata gcatgccatt
7801 aacctaggta cagaagtcca attgcttccg atctggtaaa agattcacga gatagtacct
7861 tctccgaagt aggtagagcg agtacccggc gcgtaagctc cctaattggc ccatccggca
7921 tctgtagggc gtccaaatat cgtgcctctc ctgctttgcc cggtgtatga aaccggaaag
7981 gccgctcagg agctggccag cggcgcagac cgggaacaca agctggcagt cgacccatcc
8041 ggtgctctgc actcgacctg ctgaggtccc tcagtccctg gtaggcagct ttgccccgtc
8101 tgtccgcccg gtgtgtcggc ggggttgaca aggtcgttgc gtcagtccaa catttgttgc
8161 catattttcc tgctctcccc accagctgct cttttctttt ctctttcttt tcccatcttc
8221 agtatattca tcttcccatc caagaacctt tatttcccct aagtaagtac tttgctacat
8281 ccatactcca tccttcccat cccttattcc tttgaacctt tcagttcgag ctttcccact
8341 tcatcgcagc ttgactaaca gctaccccgc ttgagcagac atcacccggg gctgaggact
8401 taattaaacc ctcagcggta ccgagctcga attcactggc cgtcgtttta caacgtcgtg
8461 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
8521 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
8581 atggcgaatg gcgccgctgt tgcccgtctc actggtgaaa agaaaaacca ccccagtaca
8641 ttaaaaacgt ccgcaatgtg ttattaagtt gtctaagcgt caatttgttt acaccacaat
8701 atatcctgc
//

                     

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Dette sted blev sidst opdateret 01. August 2010