Difference between revisions of "Part:BBa K2491030:Design"

 
(→‎Design Notes)
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===Design Notes===
 
===Design Notes===
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The RK2 region composed of oriV and trfA was synthetized. The source of the sequence for this region was GenBank: AF139061.1. A sequence encompassing the VF2 primer region together with a terminator and a prefix was added. Once the fragment was synthetized, it was linearized with the restriction enzymes NheI and EcoRI.  This fragment was ligated with the 1364 bp kanamycin resistance cassette [biobrick part BBa_I20260, 919 bp, cloned in pSB3K3, position 18A on 2017 iGEM Biodistribution plate] linearized with the restriction enzymes PstI/NheI. The RFP reporter gene [biobrick part BBa_J04450 cloned in pSB3T5, position 8D on 2017 iGEM Biodistribution plate] was added as an EcoRI/PstI fragment.  The clones were checked by restriction enzyme digestion and by sequencing.
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===Source===
 
===Source===

Revision as of 03:33, 24 October 2017


RK2 Broad Host Range Vector with Kanamycin Resistance


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found at 1508
    Illegal EcoRI site found at 3141
    Illegal XbaI site found at 3156
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 1508
    Illegal EcoRI site found at 3141
    Illegal NheI site found at 1384
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NotI site found at 9
    Illegal NotI site found at 1514
    Illegal NotI site found at 3147
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 1508
    Illegal EcoRI site found at 3141
    Illegal XhoI site found at 177
    Illegal XhoI site found at 1020
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found at 1508
    Illegal suffix found at 2
    Illegal EcoRI site found at 3141
    Illegal XbaI site found at 3156
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found at 1508
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 3141
    Illegal XbaI site found at 1523
    Illegal XbaI site found at 3156
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NgoMIV site found at 1552
    Illegal NgoMIV site found at 2143
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.


Design Notes

The RK2 region composed of oriV and trfA was synthetized. The source of the sequence for this region was GenBank: AF139061.1. A sequence encompassing the VF2 primer region together with a terminator and a prefix was added. Once the fragment was synthetized, it was linearized with the restriction enzymes NheI and EcoRI. This fragment was ligated with the 1364 bp kanamycin resistance cassette [biobrick part BBa_I20260, 919 bp, cloned in pSB3K3, position 18A on 2017 iGEM Biodistribution plate] linearized with the restriction enzymes PstI/NheI. The RFP reporter gene [biobrick part BBa_J04450 cloned in pSB3T5, position 8D on 2017 iGEM Biodistribution plate] was added as an EcoRI/PstI fragment. The clones were checked by restriction enzyme digestion and by sequencing.

Source

Rk2 Vector

References