LOCUS       pLV-EF1A-CD19.8h        8918 bp DNA     circular SYN 23-MAR-2023
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 8918)
  AUTHORS   Michele Bernasconi
  TITLE     Direct Submission
  JOURNAL   Exported Mar 30, 2023 from SnapGene 6.2.1
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8918
                     /mol_type="other DNA"
                     /organism="recombinant plasmid"
     promoter        1..229
                     /label=RSV  promoter
                     /note="/application_notes=Strong promoter; drives 
                     transcriptionof viral RNA in packaging cells."
                     /note="/description=Rous sarcoma virus enhancer/promoter"
                     /note="/full_name=Rous sarcoma virus (RSV) 
                     enhancer/promoter"
                     /note="/official_designation=RSV"
                     /note="/uuid=3f2ddca7f0cb4015b6abcec8a25c834e"
                     /note="color: #ef6cdf; direction: RIGHT"
     LTR             230..410
                     /label=5' LTR-Delta-U3
                     /note="/application_notes=Allows transcription of viral RNA
                     andits packaging into virus."
                     /note="/description=Truncated HIV-1 5' long terminal 
                     repeat"
                     /note="/full_name=HIV-1 truncated 5' LTR"
                     /note="/official_designation=Delta5' LTR"
                     /note="/uuid=44156a3083604321be44066133e8ecb9"
                     /note="color: #c54b7c; direction: RIGHT"
     misc_feature    521..565
                     /label=Psi
                     /label=nonstandard type:  Miscellaneous
                     /note="/application_notes=Allows packaging of viral RNA 
                     intovirus."
                     /note="/description=HIV-1 packaging signal"
                     /note="/full_name=HIV-1 psi packaging signal"
                     /note="/official_designation=Psi"
                     /note="/uuid=284899413ff943a99f8210a11ad8c54e"
                     /note="color: #d05c0a; direction: RIGHT"
     misc_feature    566..930
                     /locus_tag="gag"
                     /label=gag
                     /note="color: #a6acb3"
     misc_feature    1075..1308
                     /label=RRE
                     /label=nonstandard type:  Miscellaneous
                     /note="/application_notes=Rev protein binding site that 
                     allowsRev-dependent nuclear export of viral RNA during 
                     viral packaging."
                     /note="/description=HIV-1 Rev response element"
                     /note="/full_name=HIV-1 Rev response element"
                     /note="/official_designation=RRE"
                     /note="/uuid=df07099e49674cf3aba3661b54e28bd7"
                     /note="color: #fd3434; direction: RIGHT"
     CDS             1493..1537
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /note="color: #cc99b2; direction: RIGHT"
     misc_feature    1797..1802
                     /label=NheI
                     /note="color: #c0c0c0"
     misc_feature    1803..1920
                     /label=cPPT
                     /label=nonstandard type:  Miscellaneous
                     /note="/application_notes=Facilitates the nuclear import of
                     HIV-1cDNA through a central DNA flap."
                     /note="/description=Central polypurine tract"
                     /note="/full_name=Central polypurine tract"
                     /note="/official_designation=cPPT"
                     /note="/uuid=f7cb29985f9f41a7a5924d3462266f42"
                     /note="color: #46c6ef; direction: RIGHT"
     protein_bind    1938..1958
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
                     /note="color: #31849b; direction: RIGHT"
     misc_feature    1959..1964
                     /label=XbaI
                     /note="color: #a6acb3"
     promoter        1965..2176
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
                     /note="color: #ffffff; direction: RIGHT"
     primer_bind     2065..2084
                     /label=pSL-1F
                     /note="color: #------; direction: RIGHT"
     primer_bind     2065..2084
                     /label=pSL-1F
                     /note="color: black; sequence: TGATGTCGTGTACTGGCTCC; added:
                     2021-01-08"
     primer_bind     2133..2208
                     /label=CPF01
                     /note="color: black; sequence: 
                     ccgtgaacgttctttttcgcaacgggtttgccgccagaacacagggatcccaagtttgt
                     acaaaaaagcaggctgc; added: 2020-10-29"
     misc_feature    2177..2182
                     /label=BamHI
                     /note="color: #a6acb3"
     protein_bind    2182..2206
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
                     /note="color: #008080"
     misc_feature    2207..2212
                     /label=Kozak
                     /label=nonstandard type:  Miscellaneous
                     /note="/application_notes=Facilitates translation 
                     initiation ofATG start codon downstream of the Kozak 
                     sequence."
                     /note="/description=Kozak translation initiation sequence"
                     /note="/full_name=Kozak"
                     /note="/official_designation=Kozak"
                     /note="/uuid=5f18b25a1234443494a618363f09f052"
                     /note="color: #c0c0c0"
     CDS             2213..2266
                     /codon_start=1
                     /gene="CD28"
                     /locus_tag="T-cell-specific surface glycoprotein 
                     CD28isoform 1 precursor"
                     /product="T-cell-specific surface glycoprotein CD28 
                     isoform1 precursor"
                     /label=Signal peptide CD28
                     /label=CD28 signaling peptide
                     /label=T-cell-specific surface glycoprotein CD28 isofo
                     /label=T-cell-specific surface glycoprotein CD28 isoform 1 
                     precursor
                     /note="isoform 1 precursor is encoded by transcript 
                     variant1; CD28 antigen"
                     /note="color: #993366"
                     /db_xref="GI:5453611"
                     /db_xref="CCDS:CCDS2361.1"
                     /db_xref="GeneID:940"
                     /db_xref="HGNC:HGNC:1653"
                     /db_xref="HPRD:01727"
                     /db_xref="MIM:186760"
                     /protein_id="NP_006130.1"
                     /translation="MLRLLLALNLFPSIQVTG"
     CDS             2267..2272
                     /codon_start=1
                     /label=XhoI
                     /note="color: #c0c0c0"
                     /translation="LE"
     CDS             2273..2302
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /note="color: #00ff00"
                     /translation="EQKLISEEDL"
     primer_bind     complement(2284..2301)
                     /label=pHEN-rev
                     /note="color: black; sequence: AGATCCTCTTCTGAGATG; added: 
                     2021-01-08"
     CDS             2303..2332
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /note="color: #00ff00"
                     /translation="EQKLISEEDL"
     primer_bind     complement(2311..2373)
                     /label=CPR01
                     /note="color: black; sequence: 
                     gtctgtgtcatctggatgtcggCGTCTCcGTCGACCTCGAGcagatcttcttctgaaat
                     gagc; added: 2020-10-29"
     primer_bind     2317..2380
                     /label=CPF02
                     /note="color: black; sequence: 
                     ttcagaagaagatctgCTCGAGGTCGACgGAGACGccgacatccagatgacacagacta
                     catcc; added: 2020-10-30"
     CDS             2333..2338
                     /codon_start=1
                     /label=XhoI
                     /note="color: #c0c0c0"
                     /translation="LE"
     CDS             2339..2344
                     /codon_start=1
                     /label=SalI
                     /note="color: #c0c0c0"
                     /translation="VD"
     CDS             2345..2353
                     /codon_start=1
                     /label=Esp3I filler
                     /note="color: #c0c0c0"
                     /translation="GDA"
     CDS             2354..3076
                     /codon_start=1
                     /label=FMC63 scFv
                     /label=FMC63-28Z receptor protein
                     /note="color: #ffcc00"
                     /translation="DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVK
                     LLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKL
                     EITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIR
                     QPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAK
                     HYYYGGSYAMDYWGQGTSV"
     misc_feature    2435..2455
                     /label=IDT optimization
                     /note="color: #------"
     primer_bind     3049..3073
                     /label=Genom DNA CD19 - For
                     /note="color: black; sequence: GGACTACTGGGGTCAAGGAACCTCA; 
                     added: 2021-01-08"
     primer_bind     3062..3138
                     /label=CPF03
                     /note="color: black; sequence: 
                     caaggaacctcagtcaccgtctccGGTACCaccacgacgccagcgccaactattgc; 
                     added: 2020-10-29"
     primer_bind     complement(3062..3129)
                     /label=CPR02
                     /note="color: black; sequence: 
                     ggtcgcggcgctggcgtcgtggtGGTACCggagacggtgactgaggttccttg; 
                     added: 2021-01-08"
     primer_bind     complement(3062..3114)
                     /label=CPR02a
                     /note="color: black; sequence: 
                     ggccgaggagcgggcgttgtggtGGTACCggagacggtgactgaggttccttg; 
                     added: 2020-10-30"
     primer_bind     3062..3111
                     /label=CPF03a
                     /note="color: black; sequence: 
                     caaggaacctcagtcaccgtctccGGTACCACCACAACGCCCGCTCCTCG; added: 
                     2020-10-29"
     primer_bind     complement(3062..3092)
                     /label=CPR02b
                     /note="color: black; sequence: 
                     GGAGGAGGATACATAACTTCAATGGTACCggagacggtgactgaggttccttg; 
                     added: 2021-01-08"
     CDS             3077..3085
                     /codon_start=1
                     /label=Esp3I filler 2
                     /note="color: #c0c0c0"
                     /translation="TVS"
     CDS             3086..3091
                     /codon_start=1
                     /label=KpnI
                     /note="color: #c0c0c0"
                     /translation="GT"
     CDS             3092..3226
                     /codon_start=1
                     /label=CD8 Hinge
                     /note="color: #ccffff"
                     /translation="TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD
                     "
     misc_feature    3092..3193
                     /label=IDT optimization
                     /note="color: #------"
     CDS             3227..3235
                     /codon_start=1
                     /label=BpiI filler (A)
                     /note="color: #c0c0c0"
                     /translation="RRP"
     CDS             3236..3331
                     /codon_start=1
                     /locus_tag="CD28TM"
                     /label=CD28TM
                     /note="color: #ccffcc"
                     /translation="PSKPFWVLVVVGGVLACYSLLVTVAFIIFWVR"
     primer_bind     complement(3305..3351)
                     /label=CPR03d
                     /note="color: black; sequence: 
                     agtttctttctgccccgtttcctcacccagaaaataataaaggccac; added: 
                     2020-10-30"
     primer_bind     complement(3305..3332)
                     /label=CPR03c
                     /note="color: black; sequence: 
                     tgcaggagcctgctcctcttactcctcacccagaaaataataaaggccac; added: 
                     2020-10-29"
     primer_bind     3312..3353
                     /label=CPF04d
                     /note="color: black; sequence: 
                     ttattattttctgggtgaggaaacggggcagaaagaaactcc; added: 
                     2020-10-29"
     primer_bind     3312..3332
                     /label=CPF04c
                     /note="color: black; sequence: 
                     ttattattttctgggtgaggagtaagaggagcaggctcctg; added: 
                     2020-10-29"
     CDS             3332..3457
                     /codon_start=1
                     /product="41BB CSD"
                     /label=41BB
                     /note="color: #0000ff"
                     /translation="KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL"
     primer_bind     3332..3353
                     /label=CPF04b
                     /note="color: black; sequence: 
                     actggttatcaccctttactgcaaacggggcagaaagaaactcc; added: 
                     2020-10-29"
     primer_bind     3332..3353
                     /label=CD3koF
                     /note="color: black; sequence: AAACGGGGCAGAAAGAAACTCC; 
                     added: 2021-01-08"
     primer_bind     complement(3443..3472)
                     /label=A2
                     /note="color: black; sequence: 
                     cctgctcctcttactCAGttcacatcctcc; added: 2021-01-08"
     primer_bind     3448..3474
                     /label=B1
                     /note="color: black; sequence: atgtgaaCTGagtaagaggagcaggct;
                     added: 2021-01-08"
     primer_bind     complement(3457..3480)
                     /label=CPR03c
                     /note="color: black; sequence: 
                     tgcaggagcctgctcctcttactcctcacccagaaaataataaaggccac; added: 
                     2020-10-29"
     primer_bind     3457..3478
                     /label=CPF04c
                     /note="color: black; sequence: 
                     ttattattttctgggtgaggagtaagaggagcaggctcctg; added: 
                     2020-10-29"
     CDS             3458..3577
                     /codon_start=1
                     /label=CD28
                     /note="color: #33cccc"
                     /translation="SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS"
     primer_bind     3458..3478
                     /label=CPF04
                     /note="color: black; sequence: 
                     actggttatcaccctttactgcagtaagaggagcaggctcctg; added: 
                     2020-10-29"
     CDS             3578..3583
                     /codon_start=1
                     /label=Kpn2I
                     /note="color: #c0c0c0"
                     /translation="SG"
     CDS             3584..3925
                     /codon_start=1
                     /label=CD3z isoform 1 - no Esp3I
                     /note="color: #ff0000"
                     /translation="LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPE
                     MGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD
                     ALHMQALPPR"
     misc_feature    3700
                     /label=a->g
                     /note="a to g mutated to eliminate Esp3I site"
                     /note="color: #ffff00"
     primer_bind     complement(3916..3981)
                     /label=CPR04
                     /note="color: black; sequence: 
                     tcttccacgtcgcctgcttgctttaacagagagaagttcgtggccgtacgTCCGGAgcg
                     agggggc; added: 2020-10-29"
     CDS             3926..3931
                     /codon_start=1
                     /label=Kpn2I
                     /note="color: #a6acb3"
                     /translation="SG"
     CDS             3932..3937
                     /codon_start=2
                     /label=BsiWI
                     /note="color: #c0c0c0"
                     /translation="RT"
     CDS             3938..3994
                     /codon_start=1
                     /locus_tag="P2A"
                     /label=P2A Signal_peptide
                     /label=nonstandard type: Signal_peptide
                     /note="color: #ff9900"
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     complement(3962..3983)
                     /label=pSL-2R
                     /note="Sequencing with this primer did not work. 28.8.2019
                     Probably the GGG stretch is too long...
                     "
                     /note="color: #------; direction: LEFT"
     primer_bind     complement(3962..3983)
                     /label=Genom DNA pSL2R
                     /note="color: black; sequence: TTTCTTCCACGTCGCCTGCTTG; 
                     added: 2021-01-08"
     primer_bind     complement(3962..3983)
                     /label=pSL-2R
                     /note="color: black; sequence: TTTCTTCCACGTCGCCTGCTTG; 
                     added: 2021-01-08"
     CDS             3992..4711
                     /codon_start=1
                     /label=EGFP after P2A
                     /note="color: #------"
                     /translation="PVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             3995..4711
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /note="This feature has 2 segments:
                        1: 3995 .. 3997 / #05fd14 / 1a
                        2: 3998 .. 4711 / #05fd14"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(4032..4056)
                     /label=GenRev
                     /note="color: black; sequence: TCGCCGTCCAGCTCGACCAGGATGG; 
                     added: 2021-01-08"
     primer_bind     complement(4034..4050)
                     /label=PEN1-737R
                     /note="color: black; sequence: TCCAGCTCGACCAGGAT; added: 
                     2021-01-08"
     primer_bind     complement(4067..4085)
                     /label=EGFP-N-R+33
                     /note="color: black; sequence: CGGACACGCTGAACTTGTG; added: 
                     2021-01-08"
     primer_bind     complement(4100..4118)
                     /label=EGFP-N-rev
                     /note="color: black; sequence: GCTTGCCGTAGGTGGCATC; added: 
                     2021-01-08"
     primer_bind     4618..4636
                     /label=EGFP-C-F-31
                     /note="color: black; sequence: CAAAGACCCCAACGAGAAG; added: 
                     2021-01-08"
     primer_bind     4627..4643
                     /label=EGFP-for
                     /note="color: black; sequence: CAACGAGAAGCGCGATC; added: 
                     2021-01-08"
     primer_bind     4649..4666
                     /label=EGFP-C-for
                     /note="color: black; sequence: GTCCTGCTGGAGTTCGTG; added: 
                     2021-01-08"
     protein_bind    complement(4721..4745)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
                     /note="color: #31849b; direction: LEFT"
     misc_feature    4759..5356
                     /label=WPRE
                     /label=nonstandard type:  Miscellaneous
                     /note="/application_notes=Enhances virus stability in 
                     packagingcells, leading to higher titer of packaged virus; 
                     enhances higher expression of transgenes."
                     /note="/description=Woodchuck hepatitis virus 
                     posttranscriptionalregulatory element"
                     /note="/full_name=Woodchuck hepatitis virus 
                     posttranscriptionalregulatory element"
                     /note="/official_designation=WPRE"
                     /note="/uuid=0aedff8308474b369c622433538d7836"
                     /note="color: #e4b930; direction: RIGHT"
     primer_bind     complement(4830..4849)
                     /label=WHV-5R
                     /note="color: black; sequence: AGCAGCGTATCCACATAGCG; added:
                     2021-01-08"
     CDS             complement(5234..5245)
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /note="color: #cc99b2; direction: LEFT
                     Cleavage site after base 5233"
     LTR             5438..5672
                     /label=3' LTR-Delta-U3
                     /note="/application_notes=Allows packaging of viral RNA 
                     intovirus; self-inactivates the 5' LTR by a copying 
                     mechanism during viral genome integration; contains 
                     polyadenylation signal for transcription termination."
                     /note="/description=Truncated HIV-1 3' long terminal 
                     repeat"
                     /note="/full_name=HIV-1 truncated 3' LTR"
                     /note="/official_designation=DeltaU3/3' LTR"
                     /note="/uuid=c958755414c34372b909e20e81ebcef6"
                     /note="color: #05696d; direction: RIGHT"
     primer_bind     complement(5744..5761)
                     /label=EGFP-C-Rev
                     /note="color: black; sequence: AGCTGCAATAAACAAGTT; added: 
                     2021-01-08"
     polyA_signal    5745..5879
                     /label=SV40 early pA
                     /note="/application_notes=Allows transcription termination 
                     andpolyadenylation of mRNA transcribed by Pol II RNA 
                     polymerase."
                     /note="/description=Simian virus 40 early polyadenylation 
                     signal"
                     /note="/full_name=SV40 early polyadenation signal"
                     /note="/official_designation=SV40 early pA"
                     /note="/uuid=2fcb13a62e704d8095b188d9625a6a94"
                     /note="color: #f54c6b; direction: RIGHT"
     primer_bind     5781..5802
                     /label=pCI-rev
                     /note="color: black; sequence: GCAATAGCATCACAAATTTCAC; 
                     added: 2021-01-08"
     primer_bind     5835..5854
                     /label=EBV-rev
                     /note="color: black; sequence: GTGGTTTGTCCAAACTCATC; added:
                     2021-01-08"
     primer_bind     5889..5908
                     /label=SV40-for
                     /note="color: black; sequence: GCCCCTAACTCCGCCCATCC; added:
                     2021-01-08"
     rep_origin      5905..6040
                     /label=SV40 ori
                     /note="SV40 origin of replication"
                     /note="color: #ffff00"
     primer_bind     5910..5926
                     /label=SV40-for
                     /note="color: black; sequence: GCCCCTAACTCCGCCCATCC; added:
                     2021-01-08"
     primer_bind     complement(6060..6079)
                     /label=T7
                     /note="color: black; sequence: TAATACGACTCACTATAGGG; added:
                     2021-01-08"
     promoter        complement(6061..6079)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /note="color: #ffffff; direction: LEFT"
     primer_bind     complement(6061..6079)
                     /label=Short-T7
                     /note="color: black; sequence: TAATACGACTCACTATAGG; added: 
                     2021-01-08"
     primer_bind     complement(6089..6105)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: LEFT"
     primer_bind     complement(6090..6106)
                     /label=M13
                     /note="color: black; sequence: TGTAAAACGACGGCCAG; added: 
                     2021-01-08"
     primer_bind     complement(6109..6125)
                     /label=M13-40
                     /note="color: black; sequence: GTTTTCCCAGTCACGAC; added: 
                     2021-01-08"
     primer_bind     complement(6138..6157)
                     /label=pUCM13-52
                     /note="color: black; sequence: GCTGCAAGGCGATTAAGTTG; added:
                     2021-01-08"
     rep_origin      6247..6702
                     /locus_tag="F1 ori"
                     /label=F1 ori
                     /note="color: #ffff00"
     misc_feature    6704..6709
                     /label=Kozak
                     /note="color: #a6acb3"
     promoter        6728..6832
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: RIGHT"
     CDS             6833..7693
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="This feature has 2 segments:
                        1: 6833 .. 6901 / #ccffcc / signal sequence
                        2: 6902 .. 7693 / #ccffcc
                     Cleavage site after base 6901"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6878..6897)
                     /label=AmpStart
                     /note="color: black; sequence: AAAACAGGAAGGCAAAATGC; added:
                     2021-01-08"
     primer_bind     7624..7643
                     /label=AmpStop
                     /note="color: black; sequence: TCAGGCAACTATGGATGAAC; added:
                     2021-01-08"
     rep_origin      7864..8452
                     /direction=RIGHT
                     /label=pUC ori
                     /note="/application_notes=Facilitates plasmid replication 
                     in E.coli; regulates high-copy plasmid number (500-700)."
                     /note="/description=pUC origin of replication"
                     /note="/full_name=pUC origin of replication"
                     /note="/official_designation=pUC ori"
                     /note="/uuid=64a8cb3def9f49deb6752795c2d893a8"
                     /note="color: #ce0084"
     protein_bind    8740..8761
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
                     /note="color: #31849b"
     promoter        8776..8806
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
                     /note="This forward directional feature has 3 segments:
                        1: 8776 .. 8781 / #ffffff / -35
                        2: 8782 .. 8799 / #ffffff
                        3: 8800 .. 8806 / #ffffff / -10"
     primer_bind     8787..8806
                     /label=pUCM13-rev-157
                     /note="color: black; sequence: TGCTTCCGGCTCGTATGTTG; added:
                     2021-01-08"
     primer_bind     8810..8828
                     /label=pET-T7side
                     /note="color: black; sequence: GGGAATTGTGAGCGGATAAC; added:
                     2021-01-08"
     protein_bind    8814..8830
                     /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)."
                     /note="color: #31849b"
     primer_bind     8821..8840
                     /label=LacOp-for
                     /note="color: black; sequence: CGGATAACAATTTCACACAG; added:
                     2021-01-08"
     primer_bind     8838..8854
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: RIGHT"
     primer_bind     8838..8854
                     /label=M13 Reverse:
                     /note="color: black; sequence: CAGGAAACAGCTATGAC; added: 
                     2021-01-08"
     primer_bind     8838..8854
                     /label=M13r
                     /note="color: black; sequence: CAGGAAACAGCTATGAC; added: 
                     2021-01-08"
     promoter        8875..8893
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
                     /note="color: #ffffff; direction: RIGHT"
     primer_bind     8877..8893
                     /label=T3
                     /note="color: black; sequence: TTAACCCTCACTAAAGG; added: 
                     2021-01-08"
ORIGIN
        1 aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca
       61 tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga
      121 tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt
      181 gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg
      241 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc
      301 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg
      361 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg
      421 aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt
      481 gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg
      541 actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga
      601 attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta
      661 aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta
      721 gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga
      781 tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg
      841 atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt
      901 aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga
      961 caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc
     1021 acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc
     1081 tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct
     1141 gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag
     1201 ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca
     1261 ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg
     1321 ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa
     1381 atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa
     1441 ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga
     1501 acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa
     1561 ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat
     1621 agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt
     1681 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg
     1741 tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcgcta
     1801 gcttttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata
     1861 atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt
     1921 actagtgatt atcggatcaa ctttgtatag aaaagttgtc tagagggcag agcgcacatc
     1981 gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag
     2041 gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg
     2101 tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt
     2161 tgccgccaga acacagggat cccaagtttg tacaaaaaag caggctgcca ccatgctcag
     2221 gctgctcttg gctctcaact tattcccttc aattcaagta acaggactcg aggaacaaaa
     2281 actcatctca gaagaggatc tggaacaaaa gctcatttca gaagaagatc tgctcgaggt
     2341 cgacggagac gccgacatcc agatgacaca gactacatcc tccctgtctg cctctctggg
     2401 agacagagtc accatcagtt gcagggcaag tcaggacatc tctaagtatt tgaattggta
     2461 tcagcagaaa ccagatggaa ctgttaaact cctgatctac catacatcaa gattacactc
     2521 aggagtccca tcaaggttca gtggcagtgg gtctggaaca gattattctc tcaccattag
     2581 caacctggag caagaagata ttgccactta cttttgccaa cagggtaata cgcttccgta
     2641 cacgttcgga ggggggacta agttggaaat aacaggctcc acctctggat ccggcaagcc
     2701 cggatctggc gagggatcca ccaagggcga ggtgaaactg caggagtcag gacctggcct
     2761 ggtggcgccc tcacagagcc tgtccgtcac atgcactgtc tcaggggtct cattacccga
     2821 ctatggtgta agctggattc gccagcctcc acgaaagggt ctggagtggc tgggagtaat
     2881 atggggtagt gaaaccacat actataattc agctctcaaa tccagactga ccatcatcaa
     2941 ggacaactcc aagagccaag ttttcttaaa aatgaacagt ctgcaaactg atgacacagc
     3001 catttactac tgtgccaaac attattacta cggtggtagc tatgctatgg actactgggg
     3061 tcaaggaacc tcagtcaccg tctccggtac caccacaacg cccgctcctc ggccaccgac
     3121 gccagcgcca actattgcga gtcagcctct cagtctgcga cctgaggctt gtcgaccagc
     3181 agccggaggc gcagtgcaca cgagggggct ggacttcgcc tgtgatagaa gacctccttc
     3241 taagcccttt tgggtgctgg tggtggttgg tggagtcctg gcttgctata gcttgctagt
     3301 aacagtggcc tttattattt tctgggtgag gaaacggggc agaaagaaac tcctgtatat
     3361 attcaaacaa ccatttatga gaccagtaca aactactcaa gaggaagatg gctgtagctg
     3421 ccgatttcca gaagaagaag aaggaggatg tgaactgagt aagaggagca ggctcctgca
     3481 cagtgactac atgaacatga ctcccaggcg gcccggaccc acccgcaagc attaccagcc
     3541 ctatgcccca ccacgcgact tcgcagccta tcgctcctcc ggactgagag tgaagttcag
     3601 caggagcgca gacgcccccg cgtaccagca gggccagaac cagctctata acgagctcaa
     3661 tctaggacga agagaggagt acgatgtttt ggacaagagg cgtggccggg accctgagat
     3721 ggggggaaag ccgcagagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa
     3781 agataagatg gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa
     3841 ggggcacgat ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct
     3901 tcacatgcag gccctgcccc ctcgctccgg acgtacggcc acgaacttct ctctgttaaa
     3961 gcaagcaggc gacgtggaag aaaaccccgg tcccgtgagc aagggcgagg agctgttcac
     4021 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     4081 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     4141 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
     4201 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     4261 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     4321 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     4381 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     4441 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     4501 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     4561 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
     4621 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     4681 cactctcggc atggacgagc tgtacaagta agggccccgt acccagcttt cttgtacaaa
     4741 gtggtgataa tcgaattccg ataatcaacc tctggattac aaaatttgtg aaagattgac
     4801 tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt
     4861 gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt
     4921 gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt
     4981 gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg
     5041 gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg
     5101 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaagct
     5161 gacgtccttt ccatggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt
     5221 ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc
     5281 tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc
     5341 cgcctccccg catcgggaat tcccgcggtt cgctttaaga ccaatgactt acaaggcagc
     5401 tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca
     5461 acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc agatctgagc
     5521 ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg
     5581 agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag
     5641 acccttttag tcagtgtgga aaatctctag cagtagtagt tcatgtcatc ttattattca
     5701 gtatttataa cttgcaaaga aatgaatatc agagagtgag aggaacttgt ttattgcagc
     5761 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     5821 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggctcta
     5881 gctatcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     5941 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     6001 gagctattcc agaagtagtg aggaggcttt tttggaggcc tagggacgta cccaattcgc
     6061 cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg
     6121 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
     6181 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     6241 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     6301 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     6361 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     6421 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     6481 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     6541 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     6601 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     6661 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     6721 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     6781 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     6841 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     6901 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     6961 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     7021 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     7081 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     7141 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     7201 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     7261 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     7321 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     7381 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     7441 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     7501 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     7561 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     7621 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     7681 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     7741 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     7801 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     7861 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     7921 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     7981 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     8041 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     8101 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     8161 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     8221 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     8281 agagagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     8341 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     8401 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     8461 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     8521 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     8581 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
     8641 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     8701 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
     8761 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
     8821 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa
     8881 ccctcactaa agggaacaaa agctggagct gcaagctt
//
