LOCUS       Exported                6744 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pETM-14_DnaA_wt, complete sequence.
ACCESSION   KC816624
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6744)
  AUTHORS   Paetzold B, Carolis C, Ferrar T, Serrano L, Lluch-Senar M.
  TITLE     In situ overlap and sequence synthesis during DNA assembly
  JOURNAL   ACS Synth Biol 2 (12), 750-755 (2013)
  PUBMED    24161008
REFERENCE   2  (bases 1 to 6744)
  AUTHORS   Paetzold B.
  TITLE     Direct Submission
  JOURNAL   Submitted (21-MAR-2013) EMBL-CRG Systems Biology Research Unit, 
            Centre for Genomic Regulation, Dr. Aiguader 88, Barcelona 08003, 
            Spain
REFERENCE   3  (bases 1 to 6744)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..6744
                     /organism="Cloning vector pETM-14_DnaA_wt"
                     /mol_type="other DNA"
                     /PCR_primers="fwd_name: petm14-1, fwd_seq:
                     gggtctggaagttctgtt, rev_name: petm14-2, rev_seq: 
                     aagcttatattccccagaac"
                     /PCR_primers="fwd_name: lp2, fwd_seq:
                     cgccattaacctgatgttctgggg, rev_name: lp2, rev_seq: 
                     gggcccctggaacagaactt"
                     /db_xref="taxon:1471066"
     promoter        216..234
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    235..259
                     /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)."
     RBS             274..296
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             309..326
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             336..359
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             360..1763
                     /codon_start=1
                     /product="DnaA"
                     /label=DnaA
                     /protein_id="AHL28658.1"
                     /translation="MSLSLWQQCLARLQDELPATEFSMWIRPLQAELSDNTLALYAPNR
                     FVLDWVRDKYLNNINGLLTSFCGADAPQLRFEVGTKPVTQTPQAAVTSNVAAPAQVAQT
                     QPQRAAPSTRSGWDNVPAPAEPTYRSNVNVKHTFDNFVEGKSNQLARAAARQVADNPGG
                     AYNPLFLYGGTGLGKTHLLHAVGNGIMARKPNAKVVYMHSERFVQDMVKALQNNAIEEF
                     KRYYRSVDALLIDDIQFFANKERSQEEFFHTFNALLEGNQQIILTSDRYPKEINGVEDR
                     LKSRFGWGLTVAIEPPELETRVAILMKKADENDIRLPGEVAFFIAKRLRSNVRELEGAL
                     NRVIANANFTGRAITIDFVREALRDLLALQEKLVTIDNIQKTVAEYYKIKVADLLSKRR
                     SRSVARPRQMAMALAKELTNHSLPEIGDAFGGRDHTTVLHACRKIEQLREESHDIKEDF
                     SNLIRTLSS"
     CDS             1820..1837
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      1904..1951
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1988..2443
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     CDS             complement(2536..3351)
                     /codon_start=1
                     /product="aminoglycoside 3-phosphotransferase"
                     /label=aminoglycoside 3-phosphotransferase
                     /protein_id="AHL28659.1"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      3473..4061
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    4247..4389
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(4491..4682)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5491..6573)
                     /codon_start=1
                     /product="lactose operon repressor"
                     /label=lactose operon repressor
                     /protein_id="AHL28660.1"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(6574..6651)
                     /gene="lacI"
                     /label=lacI promoter