LOCUS       Exported                5612 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Broad host range vector vector pCVD028, complete sequence.
ACCESSION   KM017916
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5612)
  AUTHORS   Taton A, Unglaub F, Wright NE, Zeng WY, Paz-Yepez J, Brahamsha B, 
            Palenik B, Peterson TC, Haerizadeh F, Golden SS, Golden JW.
  TITLE     Broad-host-range vector system for synthetic biology and 
            biotechnology in cyanobacteria
  JOURNAL   Nucleic Acids Res. (2014) In press
  PUBMED    25074377
REFERENCE   2  (bases 1 to 5612)
  AUTHORS   Taton A, Unglaub F, Wright N, Zeng WY, Paz Yepes J, Brahamsha B, 
            Palenik B, Peterson T, Haerizadeh F, Golden SS, Golden JW.
  TITLE     Direct Submission
  JOURNAL   Submitted (17-JUN-2014) Division of Biological Sciences, University 
            of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093, USA
REFERENCE   3  (bases 1 to 5612)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..5612
                     /organism="Broad host range vector vector pCVD028"
                     /mol_type="other DNA"
                     /note="oribom-sacB-ko is a replication device for E coli 
                     with an origin and a bom site, for conjugal transfer, from 
                     pBR322, carrying a sacB gene for selection of double 
                     recombinants grown with sucrose and that can be assembled 
                     into a double recombination knock-out or knock-in plasmid A
                     point mutation was identified (G249C) in the ORF of sacB 
                     leading to the following amino acid change: E83D (Ryan 
                     Simkovsky) This device appears however functional in 
                     Anabaena sp PCC7120 and Synechococcus elongatus PCC7942"
                     /db_xref="taxon:1531886"
     misc_feature    complement(146..404)
                     /note="lacZa-part2; lacZalpha fragment upstream of EcoICRI"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    405..410
                     /label=ZraI
                     /note="ZraI"
     misc_feature    complement(408..3327)
                     /note="CVD-ECKO; device for the GC-adaptor assembly: 
                     Escherichia coli origin for KO"
     misc_feature    complement(408..428)
                     /note="G5C5; GC-adaptor for seamless assembly, flanking 
                     Escherichia coli origins for KO; synthetic"
     misc_feature    429..434
                     /label=SacI
                     /note="SacI"
     misc_feature    517..522
                     /label=EcoRV
                     /note="EcoRV"
     CDS             complement(542..1963)
                     /codon_start=1
                     /product="sacB"
                     /label=sacB
                     /note="sacB derived from Bacillus subtilis INSD accession 
                     AAA72307; 1; a point mutation was identified G249C in the 
                     ORF of sacB leading to the following amino acid change: 
                     E83D Ryan Simkovsky; this device appears however functional
                     in Anabaena sp. PCC7120 and Synechococcus elongatus PCC7942
                     Bacillus subtilis"
                     /protein_id="AII99744.1"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     regulatory      complement(1964..2269)
                     /regulatory_class="promoter"
                     /note="PsacB; derived from Bacillus subtilis INSD accession
                     AAA72307"
     misc_feature    2269..2274
                     /label=SacI
                     /note="SacI"
     oriT            complement(2331..2444)
                     /direction=LEFT
                     /note="bom; bom site of pBR322, for comjugal transfer; 
                     requires a helper plasmid such as pRL623 (or pRK2013)"
     misc_feature    2350..2490
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(2617..3290)
                     /direction=LEFT
                     /note="ori-pBR322; origin of replication of pBR322"
     rep_origin      complement(2676..3264)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    3301..3306
                     /label=MfeI
                     /note="MfeI"
     misc_feature    complement(3307..3327)
                     /note="C3G3; GC-adaptor for seamless assembly, flanking 
                     Escherichia coli origins for KO; synthetic"
     misc_feature    3325..3330
                     /label=ZraI
                     /note="ZraI"
     misc_feature    complement(3331..3395)
                     /note="lacZa-part1; lacZalpha fragment upstream of EcoICRI"
     misc_feature    3349..3354
                     /label=XbaI
                     /note="XbaI"
     primer_bind     complement(3391..3407)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      complement(3396..3518)
                     /regulatory_class="promoter"
                     /note="PlacZa; lacI-lacZ intergenic region derived from 
                     Escherichia coli including the promoter and RBS"
     regulatory      complement(3413..3433)
                     /regulatory_class="other"
                     /note="lacO; LacO; lac operator (lacO) from Invitrogen 
                     pTrcHis2"
     protein_bind    3415..3431
                     /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(3439..3469)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3484..3505
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(3734..4407)
                     /direction=LEFT
                     /note="ori-pUC19; origin of replication of pUC19"
     rep_origin      complement(3793..4381)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4552..5412)
                     /codon_start=1
                     /product="bla"
                     /label=bla
                     /note="confers ampicillin resistance; beta-lactamase; 
                     derived from pUC19"
                     /protein_id="AII99743.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5413..5517)
                     /gene="bla"
                     /label=AmpR promoter
     regulatory      complement(5413..5482)
                     /regulatory_class="promoter"
                     /note="Pbla; upstream region of the bla ORF including 
                     promoter and RBS; derived from pUC19"
     misc_feature    5543..5548
                     /label=ZraI
                     /note="ZraI"