LOCUS       Exported                7515 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pTcTAPN, complete sequence.
ACCESSION   HM162842
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7515)
  AUTHORS   Batista M, Marchini FK, Celedon PA, Fragoso SP, Probst CM, Preti H, 
            Ozaki LS, Buck GA, Goldenberg S, Krieger MA.
  TITLE     A high-throughput cloning system for reverse genetics in Trypanosoma
            cruzi
  JOURNAL   BMC Microbiol. 10 (1), 259 (2010)
  PUBMED    20942965
REFERENCE   2  (bases 1 to 7515)
  AUTHORS   Batista M, Marchini FK, Celedon PAF., Fragoso SP, Probst CM, Preti 
            H, Ozaki LS, Buck GA, Goldenberg S, Krieger MA.
  TITLE     Direct Submission
  JOURNAL   Submitted (28-APR-2010) Instituto Carlos Chagas, Professor Algacyr 
            Munhoz Mader, 3775, Curitiba, Parana 81350010, Brasil
REFERENCE   3  (bases 1 to 7515)
  AUTHORS   Batista M, Marchini FK, Celedon PAF., Fragoso SP, Probst CM, Preti 
            H, Ozaki LS, Buck GA, Goldenberg S, Krieger MA.
  TITLE     Direct Submission
  JOURNAL   Submitted (18-NOV-2010) Instituto Carlos Chagas, Professor Algacyr 
            Munhoz Mader, 3775, Curitiba, Parana 81350010, Brasil
REFERENCE   4  (bases 1 to 7515)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     On Nov 18, 2010 this sequence version replaced HM162842.1.
FEATURES             Location/Qualifiers
     source          1..7515
                     /organism="Cloning vector pTcTAPN"
                     /mol_type="other DNA"
                     /db_xref="taxon:878544"
     rep_origin      complement(3..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      659..1275
                     /regulatory_class="promoter"
                     /note="derived from Trypanosoma cruzi 18S ribosomal RNA 
                     gene promoter region"
     misc_feature    join(1282..1559,3940..4217,5025..5302)
                     /note="derived from Trypanosoma cruzi ubiquitin intergenic 
                     region"
     misc_feature    1569..2213
                     /note="tandem affinity purification tag containing two 
                     repeats of IgG binding protein A (B domain), TEV protease 
                     cleavage site and calmodulin binding peptide"
     CDS             1977..1997
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2043..2120
                     /codon_start=1
                     /product="calmodulin-binding peptide"
                     /label=CBP
                     /note="derived from skeletal muscle myosin light chain 
                     kinase; binds calmodulin with nanomolar affinity in the 
                     presence of calcium"
                     /translation="KRRWKKNFIAVSAANRFKKISSSGAL"
     protein_bind    2136..2169
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    2220..3930
                     /note="Gateway recombination cassette reading frame A 
                     containing attR sites, ccdB and chloramphenicol 
                     acetyltransferase genes"
     protein_bind    2223..2347
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        2372..2402
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             2456..3115
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             3457..3762
                     /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="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(3803..3927)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             4224..5018
                     /codon_start=1
                     /product="neomycin phosphotransferase"
                     /label=neomycin phosphotransferase
                     /protein_id="ADM88878.1"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     promoter        complement(5327..5345)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5366..5382)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5390..5406
                     /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(5414..5444)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5459..5480
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5768..6356)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6527..7387)
                     /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(7388..7492)
                     /gene="bla"
                     /label=AmpR promoter