LOCUS       Exported               13902 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Tri-shuttle cloning vector MiniTn-Puro-JCVI-1.7, complete sequence.
ACCESSION   GU593055
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13902)
  AUTHORS   Benders GA, Noskov VN, Denisova EA, Lartigue C, Gibson DG, 
            Assad-Garcia N, Chuang RY, Carrera W, Moodie M, Algire MA, Phan Q, 
            Alperovich N, Vashee S, Merryman C, Venter JC, Smith HO, Glass JI, 
            Hutchison CA III.
  TITLE     Cloning whole bacterial genomes in yeast
  JOURNAL   Nucleic Acids Res. 38 (8), 2558-2569 (2010)
  PUBMED    20211840
REFERENCE   2  (bases 1 to 13902)
  AUTHORS   Gibson DG, Benders GA, Lartigue C, Glass JI.
  TITLE     Direct Submission
  JOURNAL   Submitted (02-FEB-2010) Synthetic Biology, J. Craig Venter 
            Institute, 10355 Science Center Dr., San Diego, CA 92121, USA
REFERENCE   3  (bases 1 to 13902)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..13902
                     /organism="Tri-shuttle cloning vector
                     MiniTn-Puro-JCVI-1.7"
                     /mol_type="other DNA"
                     /db_xref="taxon:747760"
     mobile_element  complement(1..26)
                     /mobile_element_type="insertion sequence:IS256"
                     /note="inner inverted repeat"
     promoter        complement(45..63)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(84..100)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    108..124
                     /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(132..162)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    177..198
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(486..1074)
                     /direction=LEFT
                     /label=pUC19
                     /note="pUC19"
     gene            complement(1245..2105)
                     /gene="bla"
                     /label=bla
     CDS             complement(1245..2105)
                     /codon_start=1
                     /gene="bla"
                     /product="bla"
                     /label=bla
                     /note="ampicillin resistance"
                     /protein_id="ADE19333.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(2106..2210)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2236..2691)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     2833..2849
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2856..2874
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(2895..4067)
                     /codon_start=1
                     /product="IS256 transposase"
                     /label=IS256 transposase
                     /protein_id="ADE19332.1"
                     /translation="MTQVHFTLKSEEIQSIIEYSVKDDVSKNILTTVFNQLMENQRTEY
                     IQAKEYERTENRQSQRNGYYERSFTTRVGTLELKVPRTRDGHFSPTVFERYQRNEKALM
                     ASMLEMYVSGVSTRKVSKIVEELCGKSVSKSFVSSLTEQLEPMVNEWQNRLLSEKNYPY
                     LMTDVLYIKVREENRVLSKSCHIAIGITKDGDREIIGFMIQSGESEETWTTFFEYLKER
                     GLQGTELVISDAHKGLVSAIRKSFTNVSWQRCQVHFLRNIFTTIPKKNSKSFREAVKGI
                     FKFTDINLAREAKNRLIHDYIDQPKYSKACASLDDGFEDAFQYTVQGNSHNRLKSTNLI
                     ERLNQEVRRREKIIRIFPNQTSANRLIGAVLMDLHDEWIYSSRKYINFDK"
     mobile_element  4173..4197
                     /mobile_element_type="insertion sequence:IS256"
                     /note="outer inverted repeat"
     misc_feature    complement(4316..11323)
                     /label=pSmartKAN (BAC)
                     /note="pSmartKAN (BAC)"
     protein_bind    complement(4347..4380)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     terminator      4402..4431
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     rep_origin      4475..5089
                     /label=oriV
                     /note="origin of replication for the bacterial F plasmid"
     rep_origin      5165..5384
                     /label=ori2
                     /note="secondary origin of replication for the bacterial F 
                     plasmid; also known as oriS"
     CDS             5475..6230
                     /codon_start=1
                     /gene="repE"
                     /product="replication initiation protein for the bacterial 
                     F plasmid"
                     /label=repE
                     /translation="MAETAVINHKKRKNSPRIVQSNDLTEAAYSLSRDQKRMLYLFVDQ
                     IRKSDGTLQEHDGICEIHVAKYAEIFGLTSAEASKDIRQALKSFAGKEVVFYRPEEDAG
                     DEKGYESFPWFIKRAHSPSRGLYSVHINPYLIPFFIGLQNRFTQFRLSETKEITNPYAM
                     RLYESLCQYRKPDGSGIVSLKIDWIIERYQLPQSYQRMPDFRRRFLQVCVNEINSRTPM
                     RLSYIEKKKGRQTTHIVFSFRDITSMTTG"
     misc_feature    6233..6483
                     /gene="incC"
                     /label=incC
                     /note="incompatibility region of the bacterial F plasmid"
     CDS             6809..7984
                     /codon_start=1
                     /gene="sopA"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopA
                     /translation="MFRMKLMETLNQCINAGHEMTKAIAIAQFNDDSPEARKITRRWRI
                     GEAADLVGVSSQAIRDAEKAGRLPHPDMEIRGRVEQRVGYTIEQINHMRDVFGTRLRRA
                     EDVFPPVIGVAAHKGGVYKTSVSVHLAQDLALKGLRVLLVEGNDPQGTASMYHGWVPDL
                     HIHAEDTLLPFYLGEKDDVTYAIKPTCWPGLDIIPSCLALHRIETELMGKFDEGKLPTD
                     PHLMLRLAIETVAHDYDVIVIDSAPNLGIGTINVVCAADVLIVPTPAELFDYTSALQFF
                     DMLRDLLKNVDLKGFEPDVRILLTKYSNSNGSQSPWMEEQIRDAWGSMVLKNVVRETDE
                     VGKGQIRMRTVFEQAIDQRSSTGAWRNALSIWEPVCNEIFDRLIKPRWEIR"
     CDS             7984..8955
                     /codon_start=1
                     /gene="sopB"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopB
                     /translation="MKRAPVIPKHTLNTQPVEDTSLSTPAAPMVDSLIARVGVMARGNA
                     ITLPVCGRDVKFTLEVLRGDSVEKTSRVWSGNERDQELLTEDALDDLIPSFLLTGQQTP
                     AFGRRVSGVIEIADGSRRRKAAALTESDYRVLVGELDDEQMAALSRLGNDYRPTSAYER
                     GQRYASRLQNEFAGNISALADAENISRKIITRCINTAKLPKSVVALFSHPGELSARSGD
                     ALQKAFTDKEELLKQQASNLHEQKKAGVIFEAEEVITLLTSVLKTSSASRTSLSSRHQF
                     APGATVLYKGDKMVLNLDRSRVPTECIEKIEAILKELEKPAP"
     misc_feature    9028..9501
                     /gene="sopC"
                     /label=sopC
                     /note="centromere-like partitioning region of the bacterial
                     F plasmid"
     CDS             complement(9772..10587)
                     /codon_start=1
                     /gene="aph(3)-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     promoter        complement(10588..10690)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     misc_feature    10712..11110
                     /label=cos
                     /note="lambda cos site; allows packaging into phage lambda 
                     particles"
     terminator      11159..11190
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     protein_bind    complement(11209..11242)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     gene            complement(11349..12008)
                     /gene="HIS3"
                     /label=HIS3
     CDS             complement(11349..12008)
                     /codon_start=1
                     /gene="HIS3"
                     /product="HIS3"
                     /label=HIS3
                     /protein_id="ADE19331.1"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALLARGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHFL
                     ESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     promoter        complement(12009..12196)
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     rep_origin      complement(12196..12570)
                     /direction=LEFT
                     /label=ARSH4
                     /note="ARSH4"
     misc_feature    12197..12700
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     misc_feature    complement(12584..12700)
                     /label=CEN6
                     /note="CEN6"
     promoter        12703..12721
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     regulatory      12746..13068
                     /regulatory_class="promoter"
                     /note="spiralin promoter"
     CDS             13070..13669
                     /codon_start=1
                     /product="puromycin resistance protein"
                     /label=puromycin resistance protein
                     /protein_id="ADE19330.1"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"