LOCUS       Exported                8138 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Mammalian expression vector pEXPR61, complete sequence.
ACCESSION   MH225461
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8138)
  AUTHORS   Siciliano V.
  TITLE     Engineering modular intracellular protein sensor-actuator devices
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8138)
  AUTHORS   Siciliano V.
  TITLE     Direct Submission
  JOURNAL   Submitted (02-APR-2018) Biological Engineering, Massachusetts 
            Institute of Technology, 500 Tech Square, Cambridge, MA 02139, USA
REFERENCE   3  (bases 1 to 8138)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..8138
                     /organism="Mammalian expression vector pEXPR61"
                     /mol_type="other DNA"
                     /label=TRE_tight-scFv2-LD15-TEV2
                     /note="TRE_tight-scFv2-LD15-TEV2"
                     /db_xref="taxon:2171694"
     rep_origin      complement(222..810)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(981..1841)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(1842..1946)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1972..2427)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     2569..2585
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2595..2613
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    2628..3431
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     misc_feature    3510..3535
                     /label=SA
                     /note="splice acceptor site"
     protein_bind    4207..4227
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    4272..4290
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4307..4325
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4343..4361
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4379..4397
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4414..4432
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4450..4468
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        4480..4518
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     protein_bind    4558..4582
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4629..6146
                     /codon_start=1
                     /product="scFv2-LD15-TEV"
                     /label=scFv2-LD15-TEV
                     /note="chimeric protein"
                     /protein_id="AWB80849.1"
                     /translation="MQVQLKQSGPGLVHPSQSLSITCTVSGFSLTSYGVHWVRQSPGKG
                     LEWLGVIWSDGSTTYNSALKSRLNITKDNSKRQVFFKMNSLQADDTAIYYCAKEPPTTY
                     VCLWGQGTSVTVSSGGGGSGGGGSGGGGSELVMTQSPLSLPVTPGEPASISCRSSQSLV
                     HSNGITYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVG
                     VYYCSQSTHIPWTFGQGTKLEIKRAGRGGGGSGGGGSGGGGSGSLFKGPRDYNPISSTI
                     CHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLI
                     DGRDMIIIRMPKDFPPFPQKLKFREPQREERICLVTTNFQTKSMSSMVSDTSCTFPSSD
                     GIFWKHWIQTKDGQCGSPLVSTRDGFIVGIHSASNFTNTNNYFTSVPKNFMELLTNQEA
                     QQWVSGWRLNADSVLWGGHKVFMVKPEEPFQPVKEATQLMNELVYSQ"
     protein_bind    complement(6171..6195)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_site      6350..6405
                     /label=-beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal"
     primer_bind     complement(6766..6782)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6790..6806
                     /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(6814..6844)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6859..6880
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     misc_feature    7053..7889
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     promoter        complement(7919..7937)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(7958..7974)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    7982..7998
                     /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(8006..8036)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8051..8072
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."