LOCUS       Exported                8457 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector pCS2+MT-gli2PSM-HIS, complete sequence.
ACCESSION   MF435045
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8457)
  AUTHORS   Cerrizuela S, Vega-Lopez GA, Palacio MB, Aybar MJ.
  TITLE     Gli2 is required for the induction and migration of Xenopus laevis 
            neural crest
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8457)
  AUTHORS   Cerrizuela S, Vega-Lopez GA, Palacio MB, Aybar MJ.
  TITLE     Direct Submission
  JOURNAL   Submitted (05-JUL-2017) Developmental Biology, Instituto Superior de
            Investigaciones Biologicas (INSIBIO, CONICET), Chacabuco 461, San 
            Miguel de Tucuman, Tucuman T4000ILI, Argentina
REFERENCE   3  (bases 1 to 8457)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..8457
                     /organism="Cloning vector pCS2+MT-gli2PSM-HIS"
                     /mol_type="other DNA"
                     /db_xref="taxon:2052064"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     regulatory      35..51
                     /regulatory_class="promoter"
     CDS             100..4476
                     /codon_start=1
                     /gene="Gli2"
                     /product="Gli2 PSM"
                     /label=Gli2
                     /note="full length Gli2 protein coding sequence with 6 
                     point mutations in the PKA phosphorilation sites"
                     /protein_id="ATQ62673.1"
                     /translation="MEQKLISEEDLNEMEQKLISEEDLNEMEQKLISEEDLNEMEQKLI
                     SEEDLNEMEQKLISEEDLNEMESLGDLTMEQKLISEEDLNSRPLDVEHYLRSVHNSPTL
                     SMISAARGLSPAEVAHEHLKERGIYGLAPPPPGTTPTEYCHQMAFLASHASPYGELLVQ
                     SAAAGNTSHLHDYLTPMDVSRFSSPRVTPRLSRKRALSISPLSDASIDLQTMIRTSPNS
                     LVAYINNSRSSSAASGSYGHLSAGAISPAFSFPIHKPCSLSAALSQQRSLSSSFGHTPL
                     LHPSPTFASRQQGALTSANPAPPSNNSSAPDSVLNKVSSESAVSSTVNQVIHKRSKVKT
                     EEEADSVRFPQPPDHLTDLKEDLDKDECKQEPEHIYETNCHWDGCSKEFDTQDQLVHHI
                     NNDHIHGEKKEFVCRWQDCSREQKPFKAQYMLVVHMRRHTGEKPHKCTFEGCFKAYSRL
                     ENLKTHLRSHTGEKPYVCDHEGCNKAFSNASDRAKHQNRTHSNEKPYICKVPGCTKRYT
                     DPSSLRKHVKTVHGPEAHVTKKHRNDIIQKPSLPKENGDNEASAKLSGREHSDSVSRDQ
                     EHCLQTRTIKTEDNMMHQSSPGGQSSCSSEPSPYGNTNNIDSGVDVSLAWQGSLGDLFG
                     LEETSPVVDSTVSSWQRSGRPATPETQRIHSAETGTAEEREIKDNERFLLIYEPNATCQ
                     NTRLPTISANGFDVIGVPSSVLINPRAIELSMNDVTMMNQLNERRDATSSTLSSAYTSR
                     RSAGISPYFSSRRSAETSQFGGRLNNSSSADSYDPISTDASRRSAEASQHSGLPNLLNL
                     TPAQHYRLKAKYAAATGGPPPTPLPNMDRIGLRNKLSLMDGADFPLPPFRQLPVPRRCA
                     DGGGNAGLTPMYPHEIPGNNSRRAADPVRRTAGIDDKPLPRFSRFHSMNSMNTLHPPSL
                     SERRNGGLQHYTCSDGGLHRHVYSPRPPSISENVAMEAISCDADVPGGDDDLMLPDDVV
                     QYIRSQNREAPEQNLQTEYSSPARNLQSNTKSFHNNTPEQPRAPGAYLSRNFPALAECL
                     GQTANMQDNNMPVQWNEVSSGTVDVSDLPKQQFAAGNLAVVQQKQNFAQYQSFNQAPMQ
                     RAHNIMGQGQESVQRNISVNGQRFNYLQQRQQQMSQCQIVSSDFIPQQRYSQSQSMLSS
                     RAMQEGQSQISPSCNNMVERPGVHTHAAPSNTLNHQRLAVHGAPTQGFANNFSVNQDGL
                     HPPNAYTVQPQKNGLEPQQNTLGMSGQAFNHGMIQPRPPAAPHPPNRPRNIHPVHHPPY
                     MRSPHPVSELSPGQQTAEATPKRTSENTDPTPKDNNLLYYSGQIHMYEPNGNFGSGIDC
                     TVRQLPTMPSPGANQVTSTVDSQGLEHPPVIDFDAMMDDGDHSSLMSGTLSPGLLQSFS
                     QTSSRLTTPRNSLTLPSIPAGINNMAIGDMSSMLTTLAEESKFLAIMQLQHHHHHH"
     gene            100..4476
                     /gene="Gli2"
                     /label=Gli2
     sig_peptide     100..354
                     /gene="Gli2"
     CDS             103..132
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             142..171
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             181..210
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             220..249
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             259..288
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             322..351
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     sig_peptide     4456..4473
                     /gene="Gli2"
     CDS             4456..4473
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        complement(4484..4501)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4506..4701
                     /regulatory_class="polyA_signal_sequence"
     polyA_signal    4506..4640
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(4753..4771)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     regulatory      complement(4755..4771)
                     /regulatory_class="promoter"
     primer_bind     complement(4792..4808)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4816..4832
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(4840..4870)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4885..4906
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5194..5782)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5953..6813)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(6814..6918)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6944..7399)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        7473..8457
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"