The map of pCANTAB 5e, shows the control regions and genealogy of the vector. The section above the vector map (VH-Linker-VL) depicts the orientation of a hypothetical ScFv fragment cloned into pCANTAB 5e. Detailed sequence analysis of the vector follows. The sequence of pCANTAB 5e was assembled from the sequences of its constituents. The enzymes chosen for the analysis are those which we believe to have been commercially available in June, 1992. pCANTAB 5e has not been tested with all of these enzymes, and therefore the accuracy of the tables cannot be guaranteed.
Expression control region: lac promoter: -35: 2144-2149; -10: 2168-2174;
Gene 3 signal sequence: 2269-2313; protein synthesis begins at the start of the gene 3 signal sequence (with GTG at position 2269)
Gene 3 protein: 2428-3639 (two stop codons follow at bases 3640-3645)
M13 region: 3863-4336
M13 origin of replication: 4173 (base 4174 is the first base of the newly synthesized strand)
– Note: Earlier instructions (anything prior to print code XY-038-00-05, Rev. 3) indicated the M13 ori in the opposite direction. This was incorrect. The map on page 30 shows the correct orientation.
β-lactamase gene region: Promoter: -35: 131-136; -10: 154-159; Start codon (ATG): 201; Stop codon (TAA): 1059 (continued on page 32)
Plasmid origin of replication: 1819
Amber Stop Codon: 242
E Tag: 2380-2418; E Tag Sequence: GGT GCG CCG GTG CCG TAT CCG GAT CCG CTG GAA CCG CGT
Restriction Enzyme Analysis
No sites: Acc65 I, Afl II, Age I, Apa I, ApaL I, Asc I, Asu II, Ava III, Avr II, Bal I, BbrP I, Bbs I, Bcl I, Bfr I, Bgl II, Bpu1102 I, BsaB I, Bsg I, BsiW I, BssH II, Bst1107 I, BstB I, BstE II, BstX I, Bsu36 I, Ecl136 II, Eco47 III, EcoN I, EcoR V , Esp I, Hpa I, Kpn I, Mam I, Mlu I, Msc I, Mun I, Nhe I, Nru I, Nsi I, Pac I, Pme I, Pml I, PpuM I, Rsr II, Sac I, Sac II, Sau I, Sce I, Sma I, SnaB I, Spe I, Sph I, Spl I, Spo I, Srf I, Sse8387 I, Stu I, Swa I, Tth111 I, Xba I, Xcm I, Xma I
One site: Aat II (65), Acc I (2350), Afl III (1876), Ban II (4036), Bcg I (461), Bpm I (898), Bsa I (916), BsaA I (4109), BseA I (2397), Bsm I (2535), BspD I (3316), BspE I (2397), BspM II (2397), Cla I (3316), Dra II (7), Dra III (4109), Eag I (2372), Eam1105 I (983), EcoO109 I (7), EcoR I (3646), Hinc II (2350), Hind II (2350), Hind III (2235), Kas I (3807), Nar I (3807), Nco I (2327), Nde I (3512), Not I (2371), PaeR7 I (2356), PflM I (2233), Pst I (2344), Sal I (2350), Sca I (505), Sfi I (2316), SgrA I (2384), Sty I (2327), Xho I (2356)
Two sites: AlwN I (1462, 2976), Ava I (2356, 4218), BamH I (2400, 3009), Dsa I (2327, 3552), Eco57 I (301, 1349), Esp3 I (4467, 4516), Fsp I (763, 3786), Nae I (2322, 4006), NgoM I (2322, 4006), Nsp I (1876, 4481), Pvu I (616, 3766), Pvu II (2054, 3736), Xmn I (384, 3435)
pCANTAB 5e, as supplied, has a 49-bp region removed by Sfi I/Not I digestion. The following restriction sites are affected. The numbers in parentheses indicate the location of the site(s) remaining. One-site enzymes eliminated: Acc I, Eag I, Hinc II, Hind II, Nco I, PaeR7 I, Pst I, Sal I, Sty I, Xho I Two-site enzymes become one-site: Ava I (4218), Dsa I (3552), Nae I (4006), NgoM I (4006) Three-site enzymes become two-site: Ava II (624, 846), Bgl I (864, 3792), BspM I (2369, 3047), Sin I (624, 846)