LOCUS       Exported                6293 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Retrofitting vector pRetroES, complete sequence.
ACCESSION   AF397196
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6293)
  AUTHORS   Wang Z, Engler P, Longacre A, Storb U.
  TITLE     An efficient method for high-fidelity BAC/PAC retrofitting with a 
            selectable marker for mammalian cell transfection
  JOURNAL   Genome Res. 11 (1), 137-142 (2001)
  PUBMED    11156622
REFERENCE   2  (bases 1 to 6293)
  AUTHORS   Wang Z, Engler P, Longacre A, Storb U.
  TITLE     Direct Submission
  JOURNAL   Submitted (05-JUL-2001) Mol. Genet. Cell Biol., University of 
            Chicago, 920 E. 58th St., Chicago, IL 60637, USA
REFERENCE   3  (bases 1 to 6293)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6293
                     /organism="Retrofitting vector pRetroES"
                     /mol_type="genomic DNA"
                     /label=for in vivo retrofitting of BAC/PAC clones
                     /note="for in vivo retrofitting of BAC/PAC clones"
                     /db_xref="taxon:168860"
     promoter        470..498
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    506..522
                     /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)."
     CDS             561..2363
                     /codon_start=1
                     /gene="cre"
                     /product="GST-loxP-cre recombinase fusion protein"
                     /label=cre
                     /protein_id="AAK92454.1"
                     /translation="MLGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFEL
                     GLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVS
                     RIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMD
                     PMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRG
                     SRRASVGSHMPMAITSYSIHYTKLSGIPRAREHMAMANLLTVHQNLPALPVDATSDEVR
                     KNLMDMFRDRQAFSEHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGL
                     AVKTIQQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDF
                     DQVRSLMENSDRCQDIRNLAFLGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTK
                     TLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRA
                     LEGIFEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVN
                     IVMNYIRNLDSETGAMVRLLEDGD"
     gene            561..2363
                     /gene="cre"
                     /label=cre
     CDS             1212..1229
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     protein_bind    1266..1299
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     CDS             1332..2363
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="MANLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
                     LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
                     PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
                     LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
                     RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
                     DGD"
     misc_feature    1332..2360
                     /gene="cre"
                     /label=Region: cre recombinase
                     /note="Region: cre recombinase"
     terminator      2463..2510
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    2587..3048
                     /label=PGK poly(A) signal
                     /note="mouse phosphoglycerate kinase 1 polyadenylation 
                     signal"
     CDS             complement(3064..3867)
                     /codon_start=1
                     /product="PGK-neo"
                     /label=PGK-neo
                     /note="for mammalian cell selection"
                     /protein_id="AAK92455.1"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     promoter        complement(3881..4377)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     rep_origin      4486..4905
                     /label=R6K conditional replication origin
                     /note="R6K conditional replication origin"
     rep_origin      4509..4897
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     CDS             complement(5032..5892)
                     /codon_start=1
                     /product="beta-lactamase"
                     /label=beta-lactamase
                     /note="ampicillin-resistance protein"
                     /protein_id="AAK92456.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERSPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5893..5997)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6218..6234
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"