Generation of DNA vectors

on ice, Luria-Bertani (LB) medium was added, and the cells were allowed to recover for 30 min at 37°C. The cells were distributed on LB agar plates containing antibiotics and incubated overnight at 37°C.

Table 2: List of plasmids.

Name Backbone Purpose Reference

pC4-gBall-lacZ pCaSpeR 4 Promoter analysis Alf Herzig pC4-gBall2-lacZ(-ball3’) pCaSpeR 4 Promoter analysis This study pC4-gBall2-lacZ-SV40 pCaSpeR 4 Promoter analysis This study pC4-gBall2-lacZ pCaSpeR 4 Promoter analysis This study pC4-ABgBall2-lacZ pCaSpeR 4 Promoter analysis This study pC4-ABgBall2-lacZ(-ball3’) pCaSpeR 4 Promoter analysis This study pC4-ABgBall2-lacZ-SV40 pCaSpeR 4 Promoter analysis This study pC4-AgBall2-lacZ pCaSpeR 4 Promoter analysis This study pC4-BgBall2-lacZ pCaSpeR 4 Promoter analysis This study pC4-attB-gBall2-lacZ pCaSpeR 4 Promoter analysis This study pC4-attB-ABgBall2-lacZ pCaSpeR 4 Promoter analysis This study pC4-attB-BgBall2-lacZ pCaSpeR 4 Promoter analysis This study pC4-attB-ABgBall2-lacZ-SV40 pCaSpeR 4 Promoter analysis This study pC4-AgBall2-lacZ(-ball3’) pCaSpeR 4 Promoter analysis This study pC4 -AgBall2-lacZ-SV40 pCaSpeR 4 Promoter analysis This study pC4-attB-AgBall2-lacZ-SV40 pCaSpeR 4 Promoter analysis This study pUAS-attB pUAST Promoter analysis Alf Herzig pBSSK+gBall2-lacZ pBSSK Promoter analysis This study pBSSK+ABgBall2-lacZ pBSSK Promotor analysis This study

pBSSK+gBall2 pBSSK Rescue Alf Herzig

pBSKS-betagal pBSKS Promotor analysis Alf Herzig pCaSpeR4 pCaSpeR4 Promotor analysis Alf Herzig

pBSSK+gBall2-ball pBSSK Rescue This study

pC4-gBall2-ball pCaSpeR Rescue This study

pBSSK+BgBall2 pBSKS Promotor analysis This study pBSSK+BgBall2-lacZ pBSKS Promotor analysis This study pBSSK+gBall2-AgBall2 pBSKS Promotor analysis This study pBSSK+AgBall2 pBSKS Promotor analysis This study pBSSK+AgBall2-lacZ pBSKS Promotor analysis This study

2.1.11 Generation of DNA vectors

The primers used to generate DNA vectors are shown in Table 3. All constructs used in this study are shown in Table 4.

pBSSK+gBall2-ball: A ball coding sequence was amplified from a LD27410 cDNA clone with primers ball-CDS-5’SpeI and ball-CDS-3’NheI. The PCR product was

digested with SpeI and NheI and inserted into the SpeI and NheI sites of pBSSK+gBall2.

pC4-gBall2-ball: A 2618 bp fragment was excised with XhoI/ XbaI from pBSSK+gBall2-ball and inserted into the XhoI/ XbaI sites of pCaSpeR4.

pBSSK+ABgBall2: This construct was generated by PCR amplification of a 3629 bp fragment from the pBSSK+gBall2 vector with primers gBall-d-3’ and ABgBall2-5’XhoI, subsequent digest of the PCR product with XhoI and re-ligation of the restriction product.

pBSSK+ABgBall2-lacZ: A lacZ fragment with SalI and XbaI sites on both ends was inserted in the SalI and NheI sites of pBSSK+ABgBall2.

pC4-ABgBall2-lacZ: A 4110 bp fragment was cut out from pBSSK+ABgBall2-lacZ with XhoI and XbaI and inserted into the XhoI/ XbaI sites of pCaSpeR4.

pBSSK+BgBall2: This construct was generated by PCR amplification of the pBSSK+gBall2 vector with primers BgBall2-XhoI5’ and gBall2-d-3’, digestion of the product with XhoI and subsequent re-ligation.

pBSSK+BgBall2-lacZ: A lacZ fragment with SalI and XbaI sites on both ends was inserted in the SalI and NheI sites of pBSSK+BgBall2.

pC4-BgBall2-lacZ: A 3955 bp fragment was cut out from pBSSK+BgBall2-lacZ with XhoI and XbaI and inserted into the XhoI/ XbaI sites of pCaSpeR4.

pBSSK+gBall2-AgBall2: This construct was generated by PCR amplification of the pBSSK+gBall2 vector with primers AgBall2-5'BamHI and AgBall2-3'BamHI, digestion of the product with BamHI and subsequent re-ligation.

pBSSK+AgBall2: A 3515 bp fragment was amplified by PCR from pBSSK+gBall2-AgBall2 with the primer pair ABgBall2-5’XhoI and gBall2-d-3’, digested with XhoI and re-ligated.

pBSSK+AgBall2-lacZ: A lacZ fragment with SalI and XbaI sites on both ends was inserted into the SalI and NheI sites of pBSSK+AgBall2.

pBSSK+gBall2-lacZ: A lacZ fragment with SalI and XbaI sites on both ends was inserted into the SalI and NheI sites of pBSSK+gBall2.

pC4+AgBall2-lacZ: A 3996 bp fragment was cut out from pBSSK+AgBall2-lacZ with XhoI and XbaI and inserted into the XhoI/ XbaI sites of pCaSpeR4.

pC4-attB-BgBall2-lacZ: An attB fragment was generated by PCR from pUAS-attB with primers attB-PstI-for and attB-XhoI-rev. The PCR product was digested with PstI and XhoI and inserted into the PstI/ XhoI sites of the linearized pC4-BgBall2-lacZ.

pC4-attB-gBall2-lacZ: An attB fragment was generated by PCR from pUAS-attB with primers attB-PstI-for and attB-XhoI-rev. The PCR product was digested with PstI and XhoI and inserted into the PstI/ XhoI sites of the linearized pC4-gBall2-lacZ.

pC4-ABgBall2-lacZ(-ball3’): A lacZ fragment was generated by PCR from pC4-hs43-lacZ with primers bgal-BsiWI-for and bgal-XbaI-rev and subsequently digested with BsiWI and XbaI. pC4-ABgBall2-lacZ was digested with BsiWI and XbaI and the 11133 bp fragment was ligated with the lacZ PCR product.

pC4-ABgBall2-lacZ-SV40: An SV40 fragment with terminal XbaI and BamHI restriction sites (Alf Herzig) was inserted into the XbaI/ BamHI sites of pC4-ABgBall2-lacZ(-ball3’).

pC4-AgBall2-lacZ(-ball3’): A lacZ fragment was generated by PCR from pC4-hs43-lacZ with primers bgal-BsiWI-for and bgal-XbaI-rev and subsequently digested with BsiWI and XbaI. pC4-AgBall2-lacZ was digested with BsiWI and XbaI and the 11019 bp fragment was ligated with the lacZ PCR product.

pC4 -AgBall2-lacZ-SV40: An SV40 fragment with terminal XbaI and BamHI restriction sites (Alf Herzig) was inserted into the XbaI/ BamHI sites of pC4-AgBall2-lacZ(-ball3’).

pC4-attB-AgBall2-lacZ-SV40: An attB fragment was generated by PCR from pUAS-attB with primers pUAS-attB-PstI-for and pUAS-attB-XhoI-rev. The PCR product was digested with PstI and XhoI and inserted into the PstI/ XhoI sites of the linearized pC4-AgBall2-lacZ-SV40.

pC4-gBall2-lacZ(-ball3’): A lacZ fragment was generated by PCR from pC4-hs43-lacZ with primers bgal-BsiWI-for and bgal-XbaI-rev and subsequently digested with BsiWI and XbaI. pC4-gBall2-lacZ was digested with BsiWI and XbaI and the 10978 bp fragment was ligated with the lacZ PCR product.

pC4-gBall2-lacZ-SV40: An SV40 fragment with terminal XbaI and BamHI restriction sites (Alf Herzig) was inserted into the XbaI/ BamHI sites of pC4-gBall2-lacZ(-ball3’).

pC4-attB-gBall2-lacZ-SV40: An attB fragment was generated by PCR from pUAS-attB with primers pUAS-attB-PstI-for and pUAS-attB-XhoI-rev. The PCR product was digested with PstI and XhoI and inserted into the PstI/ XhoI sites of the linearized pC4-gBall2-lacZ-SV40.

pC4-attB-ABgBall2-lacZ-SV40: An attB fragment was generated by PCR from pUAS-attB with primers pUAS-attB-PstI-for and pUAS-attB-XhoI-rev. The PCR product was digested with PstI and XhoI and inserted into the PstI/ XhoI sites of the linearized pC4-ABgBall2-lacZ-SV40.

pC4-attB-ABgBall2-lacZ: An attB fragment was generated by PCR from pUAS-attB with primers attB-PstI-for and attB-XhoI-rev. The PCR product was digested with PstI and XhoI and inserted into the PstI/ XhoI sites of the linearized pC4-ABgBall2-lacZ.

2.2 Biochemistry