LOCUS pRF-HU2. 6323 bp DNA circular 4-JUL-2008
DEFINITION Complementary copy of pRF-HU2.
SOURCE
ORGANISM
COMMENT The vector can be use for One-Step construction of vectors for targeted gene
replacement in filamentous fungi.

The vector has been designed 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), 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'- GGACTTAAU - Reverse primer sequence
Gene-U3 (RB forward) 5'- GGCATTAAU - Forward primer sequence
Gene-U4 (RB reverse) 5'- GGTCTTAAU - Reverse primer sequence


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|486999364|
COMMENT VNTDBDATE|487005770|
COMMENT LSOWNER|
COMMENT VNTNAME|pRF-HU2.|
COMMENT VNTAUTHORNAME|Rasmus J.N. Frandsen|
COMMENT VNTAUTHOREML|raf@kvl.dk|
COMMENT VNTREPLTYPE|Plasmid
COMMENT VNTEXTCHREPL|Bacteria
FEATURES Location/Qualifiers
rep_origin complement(2679..3296)
/vntifkey="33"
/label=oriV
primer_bind 5901..5924
/vntifkey="28"
/label=RF-1
misc_feature complement(6033..6058)
/vntifkey="21"
/label=UCS\(LB)
misc_feature complement(3544..3569)
/vntifkey="21"
/label=UCS\(RB)
misc_feature complement(3671..3694)
/vntifkey="21"
/label=RF-2
misc_structure complement(1..22)
/vntifkey="88"
/label=LB
CDS complement(149..1298)
/vntifkey="4"
/label=TrfA
CDS complement(1597..2392)
/vntifkey="4"
/label=KanR
CDS complement(4601..5646)
/vntifkey="4"
/label=hph
terminator complement(3881..4597)
/vntifkey="43"
/label=TtrpC
misc_structure complement(3444..3466)
/vntifkey="88"
/label=RB
promoter complement(5647..6011)
/vntifkey="29"
/label=pTrpC
misc_feature 3450..7
/vntifkey="21"
/label=T-DNA
BASE COUNT 1426 a 1787 c 1609 g 1501 t
ORIGIN
1 tttacaccac aatatatcct gccaccagcc agccaacagc tccccgaccg gcagctcggc
61 acaaaatcac cactcgatac aggcagccca tcagtccggg acggcgtcag cgggagagca
121 gatccagccg gaactgaccc cacaaggccc tagcgtttgc aatgcaccag gtcatcattg
181 acccaggcgt gttccaccag gccgctgcct cgcaactctt cgcaggcttc gccgacctgc
241 tcgcgccact tcttcacgcg ggtggaatcc gatccgcaca tgaggcggaa ggtttccagc
301 ttgagcgggt acggctcccg gtgcgagctg aaatagtcga acatccgtcg ggccgtcggc
361 gacagcttgc ggtacttctc ccatatgaat ttcgtgtagt ggtcgccagc aaacagcacg
421 acgatttcct cgtcgatcag gacctggcaa cgggacgttt tcttgccacg gtccaggacg
481 cggaagcggt gcagcagcga caccgattcc aggtgcccaa cgcggtcgga cgtgaagccc
541 atcgccgtcg cctgtaggcg cgacaggcat tcctcggcct tcgtgtaata ccggccattg
601 atcgaccagc ccaggtcctg gcaaagctcg tagaacgtga aggtgatcgg ctcgccgata
661 ggggtgcgct tcgcgtactc caacacctgc tgccacacca gttcgtcatc gtcggcccgc
721 agctcgacgc cggtgtaggt gatcttcacg tccttgttga cgtggaaaat gaccttgttt
781 tgcagcgcct cgcgcgggat tttcttgttg cgcgtggtga acagggcaga gcgggccgtg
841 tcgtttggca tcgctcgcat cgtgtccggc cacggcgcaa tatcgaacaa ggaaagctgc
901 atttccttga tctgctgctt cgtgtgtttc agcaacgcgg cctgcttggc ctcgctgacc
961 tgttttgcca ggtcctcgcc ggcggttttt cgcttcttgg tcgtcatagt tcctcgcgtg
1021 tcgatggtca tcgacttcgc caaacctgcc gcctcctgtt cgagacgacg cgaacgctcc
1081 acggcggccg atggcgcggg cagggcaggg ggagccagtt gcacgctgtc gcgctcgatc
1141 ttggccgtag cttgctggac catcgagccg acggactgga aggtttcgcg gggcgcacgc
1201 atgacggtgc ggcttgcgat ggtttcggca tcctcggcgg aaaaccccgc gtcgatcagt
1261 tcttgcctgt atgccttccg gtcaaacgtc cgattcattc accctccttg cgggattgcc
1321 ccgactcacg ccggggcaat gtgcccttat tcctgatttg acccgcctgg tgccttggtg
1381 tccagataat ccaccttatc ggcaatgaag tcggtcccgt agaccgtctg gccgtccttc
1441 tcgtacttgg tattccgaat cttgccctgc acgaatacca gcgacccctt gcccaaatac
1501 ttgccgtggg cctcggcctg agagccaaaa cacttgatgc ggaagaagtc ggtgcgctcc
1561 tgcttgtcgc cggcatcgtt gcgccacatc taggtactaa aacaattcat ccagtaaaat
1621 ataatatttt attttctccc aatcaggctt gatccccagt aagtcaaaaa atagctcgac
1681 atactgttct tccccgatat cctccctgat cgaccggacg cagaaggcaa tgtcatacca
1741 cttgtccgcc ctgccgcttc tcccaagatc aataaagcca cttactttgc catctttcac
1801 aaagatgttg ctgtctccca ggtcgccgtg ggaaaagaca agttcctctt cgggcttttc
1861 cgtctttaaa aaatcataca gctcgcgcgg atctttaaat ggagtgtctt cttcccagtt
1921 ttcgcaatcc acatcggcca gatcgttatt cagtaagtaa tccaattcgg ctaagcggct
1981 gtctaagcta ttcgtatagg gacaatccga tatgtcgatg gagtgaaaga gcctgatgca
2041 ctccgcatac agctcgataa tcttttcagg gctttgttca tcttcatact cttccgagca
2101 aaggacgcca tcggcctcac tcatgagcag attgctccag ccatcatgcc gttcaaagtg
2161 caggaccttt ggaacaggca gctttccttc cagccatagc atcatgtcct tttcccgttc
2221 cacatcatag gtggtccctt tataccggct gtccgtcatt tttaaatata ggttttcatt
2281 ttctcccacc agcttatata ccttagcagg agacattcct tccgtatctt ttacgcagcg
2341 gtatttttcg atcagttttt tcaattccgg tgatattctc attttagcca tttattattt
2401 ccttcctctt ttctacagta tttaaagata ccccaagaag ctaattataa caagacgaac
2461 tccaattcac tgttccttgc attctaaaac cttaaatacc agaaaacagc tttttcaaag
2521 ttgttttcaa agttggcgta taacatagta tcgacggagc cgattttgaa accacaatta
2581 tgggtgatgc tgccaactta ctgatttagt gtatgatggt gtttttgagg tgctccagtg
2641 gcgtacgcct ggggggatgg gaggcccgcg ttagcgggcc gggagggttc gagaaggggg
2701 ggcacccccc ttcggcgtgc gcggtcacgc gcacagggcg cagccctggt taaaaacaag
2761 gtttataaat attggtttaa aagcaggtta aaagacaggt tagcggtggc cgaaaaacgg
2821 gcggaaaccc ttgcaaatgc tggattttct gcctgtggac agcccctcaa atgtcaatag
2881 gtgcgcccct catctgtcag cactctgccc ctcaagtgtc aaggatcgcg cccctcatct
2941 gtcagtagtc gcgcccctca agtgtcaata ccgcagggca cttatcccca ggcttgtcca
3001 catcatctgt gggaaactcg cgtaaaatca ggcgttttcg ccgatttgcg aggctggcca
3061 gctccacgtc gccggccgaa atcgagcctg cccctcatct gtcaacgccg cgccgggtga
3121 gtcggcccct caagtgtcaa cgtccgcccc tcatctgtca gtgagggcca agttttccgc
3181 gaggtatcca caacgccggc ggccgcggtg tctcgcacac ggcttcgacg gcgtttctgg
3241 cgcgtttgca gggccataga cggccgccag cccagcggcg agggcaacca gcccggtgag
3301 cgtcgcaaag gcgctcggtc ttgccttgct cgtcggatct ggaaccctgt ggttggcatg
3361 aacaaacaaa tggacgaacg gataaacctt ttcacgccct tttaaatatc cgattattct
3421 aataaacgct cttttctctt aggtttaccc gccaatatat cctgtcaaac actgatagtt
3481 taaactgaag gcgggaaacg acaatctgat catgagcgga gaattaaggg agtcacgaag
3541 cttgctgagg tcttaattaa tgcctcagca ctagtcgggg gatcctctag atctcgacgc
3601 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
3661 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
3721 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
3781 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
3841 atgccggcca cgatgcgtcc ggcgtagagg atcctctaga aagaaggatt acctctaaac
3901 aagtgtacct gtgcattctg ggtaaacgac tcataggaga gttgtaaaaa agtttcggcc
3961 ggcgtattgg gtgttacgga gcattcacta ggcaaccatg gttactattg tatacccatc
4021 ttagtaggaa tgattttcga ggtttatacc tacgatgaat gtgtgtcctg taggcttgag
4081 agttcaagga agaaacagtg caattatctt tgcgaaccca ggggctggtg acggaatttt
4141 catagtcaag ctatcagagt aaagaagagg agcatgtcaa agtacaatta gagacaaata
4201 tatagtcgcg tggagccaag agcggattcc tcagtctcgt aggtctcttg acgaccgttg
4261 atctgcttga tctcgtctcc cgaaaatgaa aatagactct gctaagctat tcttctgctt
4321 cgccggagcc tgaagggcgt actagggttg cgaggtccaa tgcattaatg cattgcagat
4381 gagctgtatc tggaagaggt aaacccgaaa cgcgttttat tcttgttgac atggagctat
4441 taaatcacta gaaggcactc tttgctgctt ggacaaatga acgtatctta tcgagatcct
4501 gaacaccatt tgtctcaact ccggagctga catcgacacc aacgatctta tatccagatt
4561 cgtcaagctg tttgatgatt tcagtaacgt taagtggatc ccggtcggca tctactctat
4621 tcctttgccc tcggacgagt gctggggcgt cggtttccac tatcggcgag tacttctaca
4681 cagccatcgg tccagacggc cgcgcttctg cgggcgattt gtgtacgccc gacagtcccg
4741 gctccggatc ggacgattgc gtcgcatcga ccctgcgccc aagctgcatc atcgaaattg
4801 ccgtcaacca agctctgata gagttggtca agaccaatgc ggagcatata cgcccggagc
4861 cgcggcgatc ctgcaagctc cggatgcctc cgctcgaagt agcgcgtctg ctgctccata
4921 caagccaacc acggcctcca gaagaagatg ttggcgacct cgtattggga atccccgaac
4981 atcgcctcgc tccagtcaat gaccgctgtt atgcggccat tgtccgtcag gacattgttg
5041 gagccgaaat ccgcgtgcac gaggtgccgg acttcggggc agtcctcggc ccaaagcatc
5101 agctcatcga gagcctgcgc gacggacgca ctgacggtgt cgtccatcac agtttgccag
5161 tgatacacat ggggatcagc aatcgcgcat atgaaatcac gccatgtagt gtattgaccg
5221 attccttgcg gtccgaatgg gccgaacccg ctcgtctggc taagatcggc cgcagcgatc
5281 gcatccatgg cctccgcgac cggctgcaga acagcgggca gttcggtttc aggcaggtct
5341 tgcaacgtga caccctgtgc acggcgggag atgcaatagg tcaggctctc gctgaattcc
5401 ccaatgtcaa gcacttccgg aatcgggagc gcggccgatg caaagtgccg ataaacataa
5461 cgatctttgt agaaaccatc ggcgcagcta tttacccgca ggacatatcc acgccctcct
5521 acatcgaagc tgaaagcacg agattcttcg ccctccgaga gctgcatcag gtcggagacg
5581 ctgtcgaact tttcgatcag aaacttctcg acagacgtcg cggtgagttc aggctttttc
5641 atatcgatgc ttgggtagaa taggtaagtc agattgaatc tgaaataaag ggaggaaggg
5701 cgaacttaag aaggtatgac cgggtcgttc acttaccttg cttgacaaac gcaccaagtt
5761 atcgtgcacc aagcagcaga tgataataat gtcctcgttc ctgtctgcta ataagagtca
5821 cacttcgagc gccgccgcta ctgctacaag tggggctgat ctgaccagtt gcctaaatga
5881 accatcttgt caaacgacac aaattttgtg ctcaccgcct ggacgactaa accaaaatag
5941 gcattcattg ttgacctcca ctagctccag ccaagcccaa aaagtgctcc ttcaatatca
6001 tcttctgtcg aggcctgatc atcgatgggc ccgctgagga cttaattaaa ccctcagcga
6061 gctcgaattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
6121 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
6181 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc cgctgttgcc
6241 cgtctcactg gtgaaaagaa aaaccacccc agtacattaa aaacgtccgc aatgtgttat
6301 taagttgtct aagcgtcaat ttg
//