01/08/10

pRF-HU2E vector sequence in GenBank format>

LOCUS pRF-HU2E. 8696 bp DNA circular 4-JUL-2008
DEFINITION Complementary copy of pRF-HU2E.
SOURCE
ORGANISM
COMMENT The vector has been designed for targeted (in locus) over expression of gene in
filamentous fungi. Compared to pRF-HU2 this vector contains the GAPDH
promoter from Aspergillus nidulans.

Like pRF-HU2 this vector allows for the simultaneous directional cloning of two
PCR amplicons into two unique sites of the vector, located on either site of the
hph (hygromycin B resistance gene) and pGAPDH, 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
Gene-U3 (RB forward) 5'- GGCATTAAU - Forward primer sequence
Gene-U4 (RB reverse) 5'- GGTCTTAAU - Reverse primer sequence

The U2 primer contains a AUG (= ATG) which can be used as start codon for
the gene you want to over express.

Digestion of vector prior to cloning:
1. Digest over night with PacI
2. Diges 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|486999381|
COMMENT VNTDBDATE|487005770|
COMMENT LSOWNER|
COMMENT VNTNAME|pRF-HU2E.|
COMMENT VNTAUTHORNAME|Rasmus J.N. Frandsen|
COMMENT VNTAUTHOREML|raf@kvl.dk|
COMMENT VNTREPLTYPE|Plasmid
COMMENT VNTEXTCHREPL|Bacteria
FEATURES Location/Qualifiers
rep_origin complement(2657..3274)
/vntifkey="33"
/label=oriV
primer_bind 8124..8149
/vntifkey="28"
/label=RF-3
primer_bind complement(3649..3672)
/vntifkey="28"
/label=RF-2
primer_bind 5879..5902
/vntifkey="28"
/label=RF-1
misc_feature complement(3522..3547)
/vntifkey="21"
/label=UCS\(RB)
misc_feature complement(8378..8403)
/vntifkey="21"
/label=UCS\(LB)
misc_feature 6073..8374
/vntifkey="21"
/label=PgpdA
misc_structure complement(8675..8696)
/vntifkey="88"
/label=LB
misc_structure complement(127..1276)
/vntifkey="88"
/label=TrfA
CDS complement(1575..2370)
/vntifkey="4"
/label=KanR
CDS complement(4579..5624)
/vntifkey="4"
/label=hph
terminator complement(3859..4575)
/vntifkey="43"
/label=TtrpC
misc_structure complement(3422..3444)
/vntifkey="88"
/label=RB
promoter complement(5625..5989)
/vntifkey="29"
/label=pTrpC
misc_feature 3408..8671
/vntifkey="21"
/label=T-DNA
BASE COUNT 2006 a 2400 c 2192 g 2098 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 tgctgaggtc ttaattaatg
3541 cctcagcact agtcggggga tcctctagat ctcgacgctc tcccttatgc gactcctgca
3601 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg
3661 catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc
3721 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
3781 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg
3841 cgtagaggat cctctagaaa gaaggattac ctctaaacaa gtgtacctgt gcattctggg
3901 taaacgactc ataggagagt tgtaaaaaag tttcggccgg cgtattgggt gttacggagc
3961 attcactagg caaccatggt tactattgta tacccatctt agtaggaatg attttcgagg
4021 tttataccta cgatgaatgt gtgtcctgta ggcttgagag ttcaaggaag aaacagtgca
4081 attatctttg cgaacccagg ggctggtgac ggaattttca tagtcaagct atcagagtaa
4141 agaagaggag catgtcaaag tacaattaga gacaaatata tagtcgcgtg gagccaagag
4201 cggattcctc agtctcgtag gtctcttgac gaccgttgat ctgcttgatc tcgtctcccg
4261 aaaatgaaaa tagactctgc taagctattc ttctgcttcg ccggagcctg aagggcgtac
4321 tagggttgcg aggtccaatg cattaatgca ttgcagatga gctgtatctg gaagaggtaa
4381 acccgaaacg cgttttattc ttgttgacat ggagctatta aatcactaga aggcactctt
4441 tgctgcttgg acaaatgaac gtatcttatc gagatcctga acaccatttg tctcaactcc
4501 ggagctgaca tcgacaccaa cgatcttata tccagattcg tcaagctgtt tgatgatttc
4561 agtaacgtta agtggatccc ggtcggcatc tactctattc ctttgccctc ggacgagtgc
4621 tggggcgtcg gtttccacta tcggcgagta cttctacaca gccatcggtc cagacggccg
4681 cgcttctgcg ggcgatttgt gtacgcccga cagtcccggc tccggatcgg acgattgcgt
4741 cgcatcgacc ctgcgcccaa gctgcatcat cgaaattgcc gtcaaccaag ctctgataga
4801 gttggtcaag accaatgcgg agcatatacg cccggagccg cggcgatcct gcaagctccg
4861 gatgcctccg ctcgaagtag cgcgtctgct gctccataca agccaaccac ggcctccaga
4921 agaagatgtt ggcgacctcg tattgggaat ccccgaacat cgcctcgctc cagtcaatga
4981 ccgctgttat gcggccattg tccgtcagga cattgttgga gccgaaatcc gcgtgcacga
5041 ggtgccggac ttcggggcag tcctcggccc aaagcatcag ctcatcgaga gcctgcgcga
5101 cggacgcact gacggtgtcg tccatcacag tttgccagtg atacacatgg ggatcagcaa
5161 tcgcgcatat gaaatcacgc catgtagtgt attgaccgat tccttgcggt ccgaatgggc
5221 cgaacccgct cgtctggcta agatcggccg cagcgatcgc atccatggcc tccgcgaccg
5281 gctgcagaac agcgggcagt tcggtttcag gcaggtcttg caacgtgaca ccctgtgcac
5341 ggcgggagat gcaataggtc aggctctcgc tgaattcccc aatgtcaagc acttccggaa
5401 tcgggagcgc ggccgatgca aagtgccgat aaacataacg atctttgtag aaaccatcgg
5461 cgcagctatt tacccgcagg acatatccac gccctcctac atcgaagctg aaagcacgag
5521 attcttcgcc ctccgagagc tgcatcaggt cggagacgct gtcgaacttt tcgatcagaa
5581 acttctcgac agacgtcgcg gtgagttcag gctttttcat atcgatgctt gggtagaata
5641 ggtaagtcag attgaatctg aaataaaggg aggaagggcg aacttaagaa ggtatgaccg
5701 ggtcgttcac ttaccttgct tgacaaacgc accaagttat cgtgcaccaa gcagcagatg
5761 ataataatgt cctcgttcct gtctgctaat aagagtcaca cttcgagcgc cgccgctact
5821 gctacaagtg gggctgatct gaccagttgc ctaaatgaac catcttgtca aacgacacaa
5881 attttgtgct caccgcctgg acgactaaac caaaataggc attcattgtt gacctccact
5941 agctccagcc aagcccaaaa agtgctcctt caatatcatc ttctgtcgag gcctgatcat
6001 cgatgggccc atcgatgatc aggcctcgag atctagagga tccttgcgcg cctaggcggc
6061 cgtggccagc ccgaattccc ttgtatctct acacacaggc tcaaatcaat aagaagaacg
6121 gttcgtcttt ttcgtttata tcttgcatcg tcccaaagct attggcggga tattctgttt
6181 gcagttggct gacttgaagt aatctctgca gatctttcga cactgaaata cgtcgagcct
6241 gctccgcttg gaagcggcga ggagcctcgt cctgtcacaa ctaccaacat ggagtacgat
6301 aagggccagt tccgccagct cattaagagc cagttcatgg gcgttggcat gatggccgtc
6361 atgcatctgt acttcaagta caccaacgct cttctgatcc agtcgatcat ccgctgaagg
6421 cgctttcgaa tctggttaag atccacgtct tcgggaagcc agcgactggt gacctccagc
6481 gtccctttaa ggctgccaac agctttctca gccagggcca gcccaagacc gacaaggcct
6541 ccctccagaa cgccgagaag aactggaggg gtggtgtcaa ggaggagtaa gctccttatt
6601 gaagtcggag gacggagcgg tgtcaagagg atattcttcg actctgtatt atagataaga
6661 tgatgaggaa ttggaggtag catagcttca tttggatttg ctttccaggc tgagactcta
6721 gcttggagca tagagggtcc tttggctttc aatattctca agtatctcga gtttgaactt
6781 attccctgtg aaccttttat tcaccaatga gcattggaat gaacatgaat ctgaggactg
6841 caatcgccat gaggttttcg aaatacatcc ggatgtcgaa ggcttggggc acctgcgttg
6901 gttgaattta gaacgtggca ctattgatca tccgatagct ctgcaaaggg cgttgcacaa
6961 tgcaagtcaa acgttgctag cagttccagg tggaatgtta tgatgagcat tgtattaaat
7021 caggagatat agcatgatct ctagttagct caccacaaaa gtcagacggc gtaaccaaaa
7081 gtcacacaac acaagctgta aggatttcgg cacggctacg gaagacggag aagccacctt
7141 cagtggactc gagtaccatt taattctatt tgtgtttgat cgagacctaa tacagcccct
7201 acaacgacca tcaaagtcgt atagctacca gtgaggaagt ggactcaaat cgacttcagc
7261 aacatctcct ggataaactt taagcctaaa ctatacagaa taagataggt ggagagctta
7321 taccgagctc ccaaatctgt ccagatcatg gttgaccggt gcctggatct tcctatagaa
7381 tcatccttat tcgttgacct agctgattct ggagtgaccc agagggtcat gacttgagcc
7441 taaaatccgc cgcctccacc atttgtagaa aaatgtgacg aactcgtgag ctctgtacag
7501 tgaccggtga ctctttctgg catgcggaga gacggacgga cgcagagaga agggctgagt
7561 aataagccac tggccagaca gctctggcgg ctctgaggtg cagtggatga ttattaatcc
7621 gggaccggcc gcccctccgc cccgaagtgg aaaggctggt gtgcccctcg ttgaccaaga
7681 atctattgca tcatcggaga atatggagct tcatcgaatc accggcagta agcgaaggag
7741 aatgtgaagc caggggtgta tagccgtcgg cgaaatagca tgccattaac ctaggtacag
7801 aagtccaatt gcttccgatc tggtaaaaga ttcacgagat agtaccttct ccgaagtagg
7861 tagagcgagt acccggcgcg taagctccct aattggccca tccggcatct gtagggcgtc
7921 caaatatcgt gcctctcctg ctttgcccgg tgtatgaaac cggaaaggcc gctcaggagc
7981 tggccagcgg cgcagaccgg gaacacaagc tggcagtcga cccatccggt gctctgcact
8041 cgacctgctg aggtccctca gtccctggta ggcagctttg ccccgtctgt ccgcccggtg
8101 tgtcggcggg gttgacaagg tcgttgcgtc agtccaacat ttgttgccat attttcctgc
8161 tctccccacc agctgctctt ttcttttctc tttcttttcc catcttcagt atattcatct
8221 tcccatccaa gaacctttat ttcccctaag taagtacttt gctacatcca tactccatcc
8281 ttcccatccc ttattccttt gaacctttca gttcgagctt tcccacttca tcgcagcttg
8341 actaacagct accccgcttg agcagacatc acccggggct gaggacttaa ttaaaccctc
8401 agcggtaccg agctcgaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
8461 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
8521 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg
8581 ccgctgttgc ccgtctcact ggtgaaaaga aaaaccaccc cagtacatta aaaacgtccg
8641 caatgtgtta ttaagttgtc taagcgtcaa tttgtttaca ccacaatata tcctgc
//