Question

the map of the pGPF is provided.

Below is a map for a plasmid called 'pGEP: 5 MCS 1234-281 AlwN I 28111 pUC orl GFP pGFP 3.3 kb AlveN 1 190의 3 MCS (1005-1098 Amp Pvul Aat II (1413) The sequence (5' to 3) of pGEP containing the promoter region, the 5' multiple cloning site (with a Bamil site, underlined), and the 5' end of the GFP coding sequence is as follows: 6: ACGACAGGTT TCCCGACTCG AAACCGCCCA GTGAGCOCAA COCAATTAATCTGAGTTAGE ITTGTGAGEGG ATAACAATTI CACACAGGAA ACACCTATGA CCATGATTAC CCCAAGCTTG 241 CATGCCTOCA GCTCGACTCT AGAGGATCEC COGCTACCOG TACIAAAAAAT GAGTAAACA 30İ GAAGA ACTTI TeACTCGACT TGTCCCAATī CTTCTTCA AT TAC.ATCCTCA TGTTAATCGC The GFP open reading frame was clonod as a is expressed as a fusion protein with Lac s plasmid, GFP Clearly indicate on the above sequenee the following elements lac Promoter : lacR The start codon for LacZ: LacZ# 1 The first codon of the GFP Sequence-GFP#1 (here are the first ten amino acids for GFP: M Lac Opsrator. lacQ The transcription start (+1) of the wRNA. circle the nucleotide and indicate +1 The Shinc Dalgamo scquenca SD Is the plasmid-encoded fusion protein subject to regulation by cAMP (catabolite repression)? SKGEELFTG) Explain your answer

Answer 1

Lac promoter has a sequence TTTACA-18- TATGTT. So it is present in the line 121

121 TCACTCATTA GGCACCCCAG GCTITACA TTCC GGCTOOTA TGGAA

Lac Z start codon ATG

first codon of GFP sequence is ATGAGT and further marked here

IRI TIOTGAGEGC ATAACAATTE CACACAGCAA ACACCTATCA SCATEATTAE GCCAAGETTO

..

TCLGAGEGG ATAACAATTT CACACAGGAA ACAGCTATGA CCATGATTAC GCCAAGCTT

I scanned the whole sequence and not able to find the shine dalgrano sequence may be image is not as clear but the sequence is AGGAGG which lies 8 bases upstream to the Lac Z sequence.

Lac operator sequence starts at : AATTGTG...........CTCACTCATT

Fusion protein can be regulated by the cAMP as the CAP binding sequence is there within  the operator sequence