LOCUS       Exported                7938 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pAP310, complete sequence.
ACCESSION   MF467537
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7938)
  AUTHORS   Park AS, Rubin EJ.
  TITLE     Clp-mediated Regulation of WhiB1 is Required for Cell Division in 
            Mycobacteria
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7938)
  AUTHORS   Park AS, Rubin EJ.
  TITLE     Direct Submission
  JOURNAL   Submitted (11-JUL-2017) Department of Immunology and Infectious 
            Diseases, Harvard School of Public Health, 665 Huntington Ave, 
            Boston, MA 02115, USA
REFERENCE   3  (bases 1 to 7938)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7938
                     /organism="Cloning vector pAP310"
                     /mol_type="other DNA"
                     /label=derived from Mycobacterium smegmatis str. MC2 155
                     /note="derived from Mycobacterium smegmatis str. MC2 155"
                     /db_xref="taxon:2204091"
     terminator      20..47
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     regulatory      72..377
                     /regulatory_class="promoter"
     protein_bind    247..264
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             406..1413
                     /codon_start=1
                     /product="WhiB1 (G58E)/eGFP-his"
                     /label=WhiB1
                     /protein_id="AWN08228.1"
                     /translation="MDWRHKAVCRDEDPELFFPVGNSGPALAQIADAKLVCNRCPVTTE
                     CLSWALESGQDAGVWGGMSEDERRALKRENARTKARTGVGGSEFGTVSKGEELFTGVVP
                     ILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFS
                     RYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFK
                     EDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGD
                     GPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKHHHHHH"
     misc_feature    406..657
                     /note="Region: WhiB family transcriptional regulator with 
                     mutation (R74E)"
     misc_feature    667..1410
                     /label=Region: eGFP with his tag at N terminus
                     /note="Region: eGFP with his tag at N terminus"
     CDS             1393..1410
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1429..1449
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1456..1473
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1480..1509
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     regulatory      1534..1576
                     /regulatory_class="terminator"
     primer_bind     complement(1579..1595)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      4420..4447
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             4631..5254
                     /codon_start=1
                     /product="TetR"
                     /label=TetR
                     /note="Tet repressor class B"
                     /protein_id="AWN08226.1"
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     5867..5883
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    5918..6052
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6116..6490)
                     /codon_start=1
                     /product="Ble"
                     /label=Ble
                     /note="zeocin resistance"
                     /protein_id="AWN08227.1"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     primer_bind     complement(6657..6673)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6681..6697
                     /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(6705..6735)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6750..6771
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      7109..7697
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"