pUC19 (70) was used as a backbone for the construction of pCMV-p. An 
8mer Notl linker (New England Biolabs # 1029) was cloned into the Smal site of 
pUC19. This destroys the Smal site, but generates in the process two SacII sites, 
one immediately on each side of the Notl linker. This vector was designated pN. 
To provide a polyadenylation signal, a 196 bp fragment from the SV40 genome 
(nucleotides 2533-2729) was purified from an SV40 containing vector and, following 
the addition of BamHI linkers, this fragment was cloned into the unique BamHI site 
within pN. The fragment is orientated such that RNA polymerase transcribing from 
a promoter upstream of the Notl site passing through the Notl site and into the 
SV40 fragment will encounter the SV40 late gene polyadenylation signal (the early 
gene polyadenylation signal appears in the opposite orientation). This vector was 
designated pNA. A 180 bp region of the SV40 genome containing late viral protein 
gene 16s/19s splice donor and accept signals [obtained as a XhoI-PstI fragment 
from pLl (71) was cloned into pNA to provide the appropriate signals. This vector 
was designated pNAss. The human cytomegalovirus immediate early gene 
promoter and enhancer was obtained as a 619 bp Thai fragment from pCM5029 
(72). This was cloned into the Hindi site of pUC18 and subsequently recovered as 
a BamHI/Hindlll fragment which was treated with T4 DNA polymerase prior to 
blunt end ligation into the vector pNAss. The E. coli p-galactosidase gene from 
pC4AUG (73) was excised as a 3530bp EcoRI - Xbal fragment and, following the 
addition of Notl linkers, cloned into the unique Notl site of the vector. 
The entire minigene containing the CMV promoter, lacZ gene, and SV40 
polyA were excised from pCMV-p on an SphI fragment. This fragment was treated 
with Klenow and ligated with Bell linkers. The pAdBglll vector was digested with 
Bglll and treated with calf intestinal phosphatase before the Bell linkered minigene 
was cloned in direct orientation into the Bglll site of pAdBglll. See Appendix F for 
overview. 
III.A.3. Construction of pAd5-CMV-lacZ 
The purpose of this construction was to remove the CMV promoter and lacZ 
gene from the pAdCMV-lacZ vector and introduce a fragment containing a portion 
of the CMV enhancer, the chicken b-actin promoter, the full-length CFTR minigene 
and a small portion of retroviral sequences from Mo-MLV. A brief description of 
the retroviral vector from which the CFTR gene was described is provided below. 
The backbone structure of this vector called pBA-CFTR includes an intact 
5’LTR of Moloney murine leukemia virus (Mo-MLV) with additional Mo-MLV 
sequences between the 5’ LTR and the internal promoter spanning nucleotide 146 
at the border of U5 to the natural Xhol site in the gag coding region at nucleotide 
624; see (74). The plasmid also contains wild-type Mo-MLV sequences from the 
Clal site at nucleotide 7674 (which was converted to a BamHI site with synthetic 
linkers) to the end of the 3’ LTR. Sequences containing the viral enhancer elements 
of the 3’ LTR from the PvuII site at nucleotide 7933 to the Xbal site at nucleotide 
8111 have been deleted. In addition to these sequences, there are flanking mouse 
genomic DNA and pBR322 sequences (spanning the Hindlll site to the EcoRI site). 
The initial promoter used in this vector was derived from a Xho I to Mbo I fragment 
of the chicken b-actin gene spanning nucleotides -266 to + 1 (75). The Mbol site 
was converted to a BamHI site and the modified p-actin fragment was cloned into 
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