LOCUS       Exported                6888 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pTc6HN, complete sequence.
ACCESSION   HM162846
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6888)
  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 6888)
  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 6888)
  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 6888)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     On Nov 18, 2010 this sequence version replaced HM162846.1.
FEATURES             Location/Qualifiers
     source          1..6888
                     /organism="Cloning vector pTc6HN"
                     /mol_type="other DNA"
                     /db_xref="taxon:878539"
     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..1559,3313..3590,4398..4675)
                     /note="derived from Trypanosoma cruzi ubiquitin intergenic 
                     region"
     misc_feature    1569..1586
                     /label=polyhistidine tag
                     /note="polyhistidine tag"
     CDS             1569..1586
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    1593..3303
                     /note="Gateway recombination cassette reading frame A 
                     containing attR sites, ccdB and chloramphenicol 
                     acetyltransferase genes"
     protein_bind    1596..1720
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        1745..1775
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1829..2488
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             2830..3135
                     /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(3176..3300)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             3597..4391
                     /codon_start=1
                     /product="neomycin phosphotransferase"
                     /label=neomycin phosphotransferase
                     /protein_id="ADM88882.1"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     promoter        complement(4700..4718)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4739..4755)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4763..4779
                     /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(4787..4817)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4832..4853
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5141..5729)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5900..6760)
                     /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(6761..6865)
                     /gene="bla"
                     /label=AmpR promoter