DNA sequencing

DNA sequencing reactions were carried out using Big Dye Terminator according to the ACRF Biomolecular Resource Facility (JCSMR, ANU) guidelines. Briefly, up to 20 ng of PCR product or 150 to 300 μg of purified plasmid was added to 1 µL of BigDye terminator, 3.2 pmol of the appropriate primer, and 3.5 µL of reaction buffer, and made up to a total reaction volume of 20 µL with sterile water. The details of the oligonucleotide primers used in the sequencing reactions are found in Table 2-10. The sequencing reaction was then performed as follows:

Initial denaturation - 96°C for 5 min

Amplification and detection – 30 cycles of: a. Denaturation - 96°C for 10 s b. Annealing – 50°C for 5 s c. Extension - 60°C for 4 min

Cooling – hold at 4°C

Primer Name Used to Sequence

2Lf Seq Rev pU3.2kbF; pU3.3kbF; HSV-1 ESminigB_Cre; HSV-1 minigB_Cre 2Rf Seq Fwd pU3.2kbF; pU3.3kbF; HSV-1 ESminigB_Cre; HSV-1 minigB_Cre 3Lf Seq Fwd pU3.3kbF; HSV-1 ESminigB_Cre; HSV-1 minigB_Cre

3Lf Seq Rev pU3.3kbF

3Rf Seq Rev pU3.3kbF; HSV-1 ESminigB_Cre; HSV-1 minigB_Cre 3Rf Seq Fwd pU3.3kbF

BGH Seq Fwd pU26/7 pICP47/Tdtom BGH Seq Rev pU26/7 pICP47/Tdtom

bla seq pT UL3/UL4; pT eGC; pT pgB_eGC; pT pICP6_eGC

Cre Lf Seq

pT eGC; pT pgB_eGC; pT pICP6_eGC; HSV-1 pICP47_eGC; HSV-1 pICP6_eGC; HSV-1 pgB_eGC; HSV-1 pC_eGC; pT pICP0 mC Cre; pU3.2kbF-minigB_Cre; HSV-1 ESminigB_Cre; pT pICP0_eGC; HSV-1 pICP0_eGC; HSV-1 minigB_Cre

Primer Name Used to Sequence

Cre Rf Seq

pT eGC; pT pgB_eGC; pT pICP6_eGC; HSV-1 pICP47_eGC; HSV-1 pICP6_eGC; HSV-1 pgB_eGC; pT pICP0 mC Cre; pU3.2kbF-minigB_Cre; HSV-1 ESminigB_Cre; pT pICP0_eGC; HSV-1 pICP0_eGC; HSV-1 minigB_Cre

ef-1 seq pT UL3/UL4; pT eGC; pT pgB_eGC; pT pICP6_eGC

eGFP/Cre Lf Seq

pT eGC; pT pgB_eGC; pT pICP6_eGC; HSV-1 pICP47_eGC; HSV-1 pICP6_eGC; HSV-1 pgB_eGC; HSV-1 pC_eGC; pU3.2kbF-minigB_Cre; HSV-1 ESminigB_Cre; pT pICP0_eGC; HSV-1 pICP0_eGC; HSV-1 minigB_Cre

eGFP/Cre Rf Seq

pT eGC; pT pgB_eGC; pT pICP6_eGC; HSV-1 pICP47_eGC; HSV-1 pICP6_eGC; HSV-1 pgB_eGC; HSV-1 pC_eGC; pU3.2kbF-minigB_Cre; HSV-1 ESminigB_Cre; pT pICP0_eGC; HSV-1 pICP0_eGC; HSV-1 minigB_Cre

EGFPnoMet

pU3.0.5kbF-Venus; pU3.1kbF-Venus; pU3.2kbF-Venus; pU3.3kbF- Venus; pU3.2kbF-minigB_Cre; HSV-1 ESminigB_Cre; pT pICP0_eGC; HSV-1 pICP0_eGC; HSV-1 minigB_Cre

ER no Met HSV-1 minigB_Cre FwdHSV-gBend pU26/7 pICP47/Tdtom

m13 Rev+ pU26/7 pICP47/Tdtom mCfwd pT pICP0 mC Cre

mCrev pT pICP0 mC Cre pgB R pU3.2kbF-gB_Cre pICP0 Seq Fwd HSV-1 pICP0_eGC pICP0 Seq Rev HSV-1 pICP0_eGC

pICP47 Seq Fwd HSV-1 pICP47_eGC; pU26/7 pICP47/Tdtom; pU3.0.5kbF-Venus; _{pU3.1kbF-Venus; pU3.2kbF-Venus; pU3.3kbF-Venus} pICP47 Seq Rev HSV-1 pICP47_eGC; pU26/7 pICP47/Tdtom; pU3.0.5kbF-Venus; _{pU3.1kbF-Venus; pU3.2kbF-Venus; pU3.3kbF-Venus}

pSC11lacZseq pU26/7 pICP47/Tdtom

ptracer CMV IE Rf pT UL3/UL4; pT CMV IE_mC; HSV-1 pC_mC; pT pICP0 mC Cre

ptracer mC Lf pT U1 pC_mC; pU3.2kbF-minigB_Cre; HSV-1 ESminigB_Cre; HSV-1 L3/UL4; pT eGC; pT pgB_eGC; pT pICP6_eGC; pT CMV IE_mC; HSV- minigB_Cre

ptracer UL3 Lf

pT UL3/UL4; pT eGC; pT pgB_eGC; pT pICP6_eGC; HSV-1 pC_mC; HSV-1 pICP6_eGC; HSV-1 pgB_eGC; HSV-1 pC_eGC; pU3.0.5kbF; pU3.1kbF; pU3.2kbF; pU3.3kbF; pT pICP0 mC Cre; pU3.0.5kbF-Venus; pU3.1kbF- Venus; pU3.2kbF-Venus; pU3.3kbF-Venus; pU3.2kbF-minigB_Cre; HSV-1 ESminigB_Cre; pU3.2kbF-gB_Cre; HSV-1 minigB_Cre

Primer Name Used to Sequence

ptracer UL4 Rf

pT UL3/UL4; pT eGC; pT pgB_eGC; pT pICP6_eGC; HSV-1 pC_mC; HSV-1 pICP47_eGC; HSV-1 pICP6_eGC; HSV-1 pgB_eGC; HSV-1 pC_eGC; pU3.0.5kbF; pU3.1kbF; pU3.2kbF; pU3.3kbF; pT pICP0 mC Cre; pU3.0.5kbF-Venus; pU3.1kbF-Venus; pU3.2kbF-Venus; pU3.3kbF- Venus; pU3.2kbF-minigB_Cre; HSV-1 ESminigB_Cre; pT pICP0_eGC; HSV-1 pICP0_eGC; HSV-1 minigB_Cre

pX330 Seq F pX330-ER pX330 Seq R pX330-ER

Seq RevpEGFPN1

pT eGC; pT pgB_eGC; pT pICP6_eGC; HSV-1 pgB_eGC; pU3.0.5kbF- Venus; pU3.3kbF-Venus; pU3.2kbF-minigB_Cre; HSV-1 ESminigB_Cre; pT pICP0_eGC; HSV-1 pICP0_eGC; pU3.2kbF-gB_Cre; HSV-1

minigB_Cre

Seq T7 Pro pU3.0.5kbF; pU3.1kbF; pU3.2kbF; pU3.3kbF SV40 F pU3.2kbF-gB_Cre

Tdt Fwd pU26/7 pICP47/Tdtom UL26 Seq Rev pU26/7 pICP47/Tdtom

UL27 Seq Fwd pU26/7 pICP47/Tdtom; pU3.0.5kbF; pU3.1kbF; pU3.2kbF; pU3.3kbF UL27 Seq Rev pU26/7 pICP47/Tdtom

UL3 Seq Fwd

HSV-1 pC_mC; HSV-1 pICP47_eGC; HSV-1 pICP6_eGC; HSV-1 pgB_eGC; HSV-1 pC_eGC; HSV-1 ESminigB_Cre; HSV-1 pICP0_eGC; HSV-1

minigB_Cre UL3 Seq Rev

HSV-1 pC_mC; HSV-1 pICP6_eGC; HSV-1 pgB_eGC; HSV-1 pC_eGC; pU3.0.5kbF; pU3.1kbF; pU3.2kbF; pU3.3kbF; HSV-1 ESminigB_Cre; HSV-1 pICP0_eGC; HSV-1 minigB_Cre

UL4 Seq Fwd

HSV-1 pC_mC; HSV-1 pICP47_eGC; HSV-1 pICP6_eGC; HSV-1 pgB_eGC; HSV-1 pC_eGC; pU3.0.5kbF; pU3.1kbF; pU3.2kbF; pU3.3kbF; HSV-1 ESminigB_Cre; HSV-1 pICP0_eGC

UL4 Seq Rev

HSV-1 pC_mC; HSV-1 pICP47_eGC; HSV-1 pICP6_eGC; HSV-1 pgB_eGC; HSV-1 pC_eGC; HSV-1 ESminigB_Cre; HSV-1 pICP0_eGC; HSV-1

minigB_Cre

An ethanol/sodium acetate precipitation was performed to remove excess dye terminators from the sequencing reaction. 80 μL of a solution containing 75 mM sodium acetate, 3.125 mM EDTA and 75% ethanol was added to each 20 μL sequencing reaction and incubated at room temperature for 15 minutes to precipitate the extension products. The solution was centrifuged at 20200 g for 20 minutes to pellet the extension products, and then washed with 70% ethanol. Samples were dried and submitted to the ACRF Biomolecular Resource Facility (JCSMR, ANU) for sequencing. Vector NTI (version 11.0;

Life Technologies) and Chromas (Technelysium) were used for DNA sequence analysis. Sequences were analysed by comparison to the published HSV-1 KOS genome sequence (Accession #JQ673480, Macdonald et al., 2012) and the original assembled plasmid sequence.