LOCUS       Exported                5133 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector pCRm-Rluc-PAC, complete sequence.
ACCESSION   KF035116
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5133)
  AUTHORS   Martin JL, Yates PA, Soysa R, Alfaro JF, Yang F, Burnum-Johnson KE, 
            Petyuk VA, Weitz KK, Camp DG II, Smith RD, Wilmarth PA, David LL, 
            Ramasamy G, Myler PJ, Carter NS.
  TITLE     Metabolic Reprogramming during Purine Stress in the Protozoan 
            Pathogen Leishmania donovani
  JOURNAL   PLoS Pathog. 10 (2), E1003938 (2014)
  PUBMED    24586154
REFERENCE   2  (bases 1 to 5133)
  AUTHORS   Yates PA, Soysa R, Carter NS.
  TITLE     A dual luciferase system for analysis of post-transcriptional gene 
            expression in Leishmania
  JOURNAL   Unpublished
REFERENCE   3  (bases 1 to 5133)
  AUTHORS   Yates PA, Soysa R, Carter NS.
  TITLE     Direct Submission
  JOURNAL   Submitted (13-MAY-2013) Biochemistry & Molecular Biology, Oregon 
            Health & Science University, 3181 SW Sam Jackson Park Rd, Portland, 
            OR 97239, USA
REFERENCE   4  (bases 1 to 5133)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..5133
                     /organism="Cloning vector pCRm-Rluc-PAC"
                     /mol_type="other DNA"
                     /db_xref="taxon:1391437"
     misc_feature    1..13
                     /label=SfiI restriction site
                     /note="SfiI restriction site"
     CDS             14..1597
                     /codon_start=1
                     /gene="Rluc-PAC"
                     /product="Renilla luciferase-puromycin acetyltransferase 
                     fusion protein"
                     /label=Rluc-PAC
                     /protein_id="AGU01698.1"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQTGGGSGGGGSGGGGSGGMTEYKPTVRLAT
                     RDDVPRAVRTLAAAFADYPATRHTVDPDRHIERVTELQELFLTRVGLDIGKVWVADDGA
                     AVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAAQQQMEGLLAPHRPKEPAWFLATVGV
                     SPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSAPRNLPFYERLGFTVTADVEVPEGPR
                     TWCMTRKPGA"
     gene            14..1597
                     /gene="Rluc-PAC"
                     /label=Rluc-PAC
     CDS             14..946
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     CDS             998..1597
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     misc_feature    1599..1610
                     /label=SfiI restriction site
                     /note="SfiI restriction site"
     misc_feature    1611..5133
                     /label=pCR-BluntII-TOPO
                     /note="pCR-BluntII-TOPO"
     promoter        complement(1692..1710)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1728..1744)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1752..1768
                     /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(1776..1806)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1821..1842
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(2130..2718)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(2856..3230)
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     misc_difference 3225
                     /replace="g"
                     /label=removes SfiI site
                     /note="removes SfiI site"
     regulatory      3227..3236
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             complement(3437..4231)
                     /codon_start=1
                     /gene="aph(3)-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(4580..4882)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     5020..5036
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        5043..5061
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"