LOCUS       Exported                6749 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pLR4, complete sequence.
ACCESSION   HQ007052
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6749)
  AUTHORS   Koksharov MI, Ugarova NN.
  TITLE     Thermostabilization of luciferase by in vivo directed evolution
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6749)
  AUTHORS   Koksharov MI, Ugarova NN.
  TITLE     Direct Submission
  JOURNAL   Submitted (30-JUL-2010) Department of Chemistry, Lomonosov Moscow 
            State University, Leninskie Gory, Bld. 1/11, GSP-3, Moscow 119991, 
            Russia
REFERENCE   3  (bases 1 to 6749)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6749
                     /organism="Cloning vector pLR4"
                     /mol_type="other DNA"
                     /db_xref="taxon:910449"
     CDS             complement(5..814)
                     /codon_start=1
                     /product="LuxR"
                     /label=LuxR
                     /note="bacterial activator of transcription"
                     /protein_id="ADP02962.1"
                     /translation="MIYNTQNLRQTIGKDKEMGMKDINADDTYRIINKIKACRSNNDIN
                     QCLSDMTKMVHCEYYLLAIIYPHSMVKSDISILDNYPKKWRQYYDDANLIKYDPIVDYS
                     NSNHSPINWNIFENNAVNKKSPNVIKEAKSSGLITGFSFPIHTANNGFGMLSFAHSEKD
                     NYIDSLFLHACMNIPLIVPSLVDNYRKINIANNKSNNDLTKREKECLAWACEGKSSWDI
                     SKILGCSKRTVTFHLTNAQMKLNTTNRCQSISKAILTGAIDCPYFKS"
     CDS             937..1557
                     /codon_start=1
                     /product="LuxI"
                     /label=LuxI
                     /note="bacterial autoinductor"
                     /protein_id="ADP02963.1"
                     /translation="MVCYSRLNAREVGMTIMIKKSDFLAIPSEEYKGILSLRYQVFKQR
                     LEWDLVVENNLESDEYDNSNAEYIYACDDTENVSGCWRLLPTTGDYMLKSVFPELLGQQ
                     SAPKDPNIVELSRFAVGKNSSKINNSASEITMKLFEAIYKHAVSQGITEYVTVTSTAIE
                     RFLKRIKVPCHRIGDKEIHVLGDTKSVVLSMPINEQFKKAVLN"
     CDS             1615..3285
                     /codon_start=1
                     /product="C-terminally tagged firefly luciferase"
                     /label=C-terminally tagged firefly luciferase
                     /note="Luc; derived from Luciola mingrelica firefly 
                     luciferase gene in INSD accession S61961; native C-terminal
                     peptide AKM is changed to SGPVEHHHHHH; contains 3 
                     additional restriction sites (BstBI, SalI, XhoI) within the
                     luciferase gene"
                     /protein_id="ADP02964.1"
                     /translation="MEMEKEENVVYGPLPFYPIEEGSAGIQLHKYMHQYAKLGAIAFSN
                     ALTGVDISYQEYFDITCRLAEAMKNFGMKPEEHIALCSENCEEFFIPVLAGLYIGVAVA
                     PTNEIYTLRELNHSLGIAQPTIVFSSRKGLPKVLEVQKTVTCIKKIVILDSKVNFGGHD
                     CMETFIKKHVELGFQPSSFVPIDVKNRKQHVALLMNSSGSTGLPKGVRITHEGAVTRFS
                     HAKDPIYGNQVSPGTAILTVVPFHHGFGMFTTLGYFACGYRVVMLTKFDEELFLRTLQD
                     YKCTSVILVPTLFAILNKSELIDKFDLSNLTEIASGGAPLAKEVGEAVARRFNLPGVRQ
                     GYGLTETTSAFIITPEGDDKPGASGKVVPLFKVKVIDLDTKKTLGVNRRGEICVKGPSL
                     MLGYSNNPEATRETIDEEGWLHTGDIGYYDEDEHFFIVDRLKSLIKYKGYQVPPAELES
                     VLLQHPNIFDAGVAGVPDPDAGELPGAVVVMEKGKTMTEKEIVDYVNSQVVNHKRLRGG
                     VRFVDEVPKGLTGKIDAKVIREILKKPQSGPVEHHHHHH"
     CDS             3265..3282
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      3349..3396
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     regulatory      3350..3396
                     /regulatory_class="terminator"
                     /note="T7 terminator"
     rep_origin      3433..3888
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     rep_origin      3450..3756
                     /label=f1 origin
                     /note="f1 origin"
     promoter        3915..4019
                     /gene="bla"
                     /label=AmpR promoter
     regulatory      3950..3978
                     /regulatory_class="promoter"
                     /note="amp promoter"
     CDS             4020..4880
                     /codon_start=1
                     /product="Bla"
                     /label=Bla
                     /note="beta-lactamase; ampicillin resistance protein"
                     /protein_id="ADP02965.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      5035..5654
                     /label=pBR322 origin
                     /note="pBR322 origin"
     rep_origin      5051..5639
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    5825..5965
                     /label=bom
                     /note="basis of mobility region from pBR322"
     misc_feature    complement(6067..6258)
                     /label=Region: ROP
                     /note="Region: ROP"
     CDS             complement(6067..6258)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"