LOCUS       Exported               13417 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Leishmania vector pIR3HYG-GW (a)-GFP+ (b), complete sequence.
ACCESSION   MG737713
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13417)
  AUTHORS   Brettmann EA, Lye LF, Beverley SM.
  TITLE     Spontaneous excision and facilitated recovery as a control for 
            phenotypes arising from RNA interference and other dominant 
            transgenes
  JOURNAL   Mol. Biochem. Parasitol. (2018) In press
  PUBMED    29357296
REFERENCE   2  (bases 1 to 13417)
  AUTHORS   Brettmann EA, Beverley SM, Lye L-F.
  TITLE     Direct Submission
  JOURNAL   Submitted (28-DEC-2017) Department of Molecular Microbiology, 
            Washington University School of Medicine, 660 S. Euclid Ave, Campus 
            Box 8230, St. Louis, MO 63110, USA
REFERENCE   3  (bases 1 to 13417)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Assembly Method       :: Lasergene v. 12
            Sequencing Technology :: Sanger dideoxy sequencing; 454 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..13417
                     /organism="Leishmania vector pIR3HYG-GW (a)-GFP+ (b)"
                     /mol_type="other DNA"
                     /db_xref="taxon:2079185"
     misc_feature    complement(25..149)
                     /label=attR2- attR2 recombination site
                     /note="attR2- attR2 recombination site"
     protein_bind    25..149
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(190..495)
                     /codon_start=1
                     /product="ccdB"
                     /label=ccdB
                     /note="ccdB gene for negative selection"
                     /protein_id="AUT31551.1"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     CDS             complement(837..1496)
                     /codon_start=1
                     /product="CmR"
                     /label=CmR
                     /note="Chloramphenicol resistance gene"
                     /protein_id="AUT31552.1"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     promoter        complement(1550..1580)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    complement(1605..1729)
                     /label=attR1- attR1 recombination site
                     /note="attR1- attR1 recombination site"
     protein_bind    complement(1605..1729)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     misc_feature    1741..2189
                     /label=MusPEX11- MusPEX11 loop sequences
                     /note="MusPEX11- MusPEX11 loop sequences"
     misc_feature    2200..2305
                     /label=MYC3- MYC3 loop sequences
                     /note="MYC3- MYC3 loop sequences"
     CDS             2204..2233
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2240..2269
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2276..2305
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     misc_feature    2321..2445
                     /label=attR1- attR1 recombination site
                     /note="attR1- attR1 recombination site"
     protein_bind    2321..2445
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        2470..2500
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             2554..3213
                     /codon_start=1
                     /product="CmR"
                     /label=CmR
                     /note="Chloramphenicol resistance gene"
                     /protein_id="AUT31553.1"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             3555..3860
                     /codon_start=1
                     /product="ccdB"
                     /label=ccdB
                     /note="ccdB gene for negative selection"
                     /protein_id="AUT31554.1"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     misc_feature    3901..4025
                     /label=attR2- attR2 recombination site
                     /note="attR2- attR2 recombination site"
     protein_bind    complement(3901..4025)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(4227..4245)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    4322..5043
                     /note="alpha tub IR- Leishmania braziliensis strain M2903 
                     alpha-tubulin intergenic region"
     misc_feature    4973..4974
                     /label=SA- spice acceptor AG dinucleotide
                     /note="SA- spice acceptor AG dinucleotide"
     CDS             5062..5781
                     /codon_start=1
                     /product="GFP+"
                     /label=GFP+
                     /protein_id="AUT31555.1"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     promoter        complement(5974..5992)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    6059..6607
                     /note="LPG1 IR- Leishmania donovani strain 1S2D LPG1 
                     intergenic region"
     misc_feature    6561..6562
                     /label=SA- spice acceptor AG dinucleotide
                     /note="SA- spice acceptor AG dinucleotide"
     RBS             6619..6641
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6667..7692
                     /codon_start=1
                     /product="HYG"
                     /label=HYG
                     /note="hygromycin B resistance gene"
                     /protein_id="AUT31556.1"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     misc_feature    7728..8299
                     /note="1.7K IR- Leishmania major strain LT252 1.7K 
                     intergenic region"
     misc_feature    8296..9381
                     /note="3 SSU- segment containing 3 region of Leishmania 
                     major strain Friedlin small subunit ribosomal RNA"
     misc_feature    9382..12243
                     /note="pBSII SK- Backbone sequences of the bacterial 
                     expression vector, pBluescript II SK(-), from Stratagene"
     promoter        complement(9398..9416)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(9437..9453)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    9461..9477
                     /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(9485..9515)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9530..9551
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(9839..10427)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(10598..11458)
                     /codon_start=1
                     /product="ampR"
                     /label=ampR
                     /note="ampicillin resistance marker"
                     /protein_id="AUT31557.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(11459..11563)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      11590..12045
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     12186..12202
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        12212..12230
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    12212..12228
                     /label=pT7- T7 RNA polymerase promoter
                     /note="pT7- T7 RNA polymerase promoter"
     misc_feature    12244..13156
                     /note="5 SSU- segment containing 5 region of Leishmania 
                     major strain Friedlin small subunit ribosomal RNA"
     misc_feature    13185..13417
                     /note="DST IR- Leishmania major strain DST intergenic 
                     region"
     misc_feature    13340..13341
                     /label=SA- spice acceptor AG dinucleotide
                     /note="SA- spice acceptor AG dinucleotide"