LOCUS       pLV-EF1A-CD19.8h        8885 bp DNA     circular SYN 23-MAR-2023
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 8885)
  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..8885
                     /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"
     promoter        3..229
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
                     /note="color: #ffffff; 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: #c0c0c0"
     misc_feature    1965..2176
                     /locus_tag="EFS"
                     /label=EFS
                     /note="color: #0000ff"
     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     2133..2208
                     /label=CPF01
                     /note="color: black; sequence: 
                     ccgtgaacgttctttttcgcaacgggtttgccgccagaacacagggatcccaagtttgt
                     acaaaaaagcaggctgc; added: 2020-10-29"
     misc_feature    2177..2182
                     /label=BamHI
                     /note="color: #c0c0c0"
     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: #31849b; direction: RIGHT"
     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..3958
                     /codon_start=1
                     /label=CD19.8h.8TM.28.BB.3z
                     /note="color: #------"
                     /translation="MLRLLLALNLFPSIQVTGLEEQKLISEEDLEQKLISEEDLLEVDG
                     DADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSG
                     VPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKP
                     GSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGV
                     IWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDY
                     WGQGTSVTVSGTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY
                     IWAPLAGTCGVLLLSLVITLYCSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAA
                     YRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELSGLRVKFSRSADAP
                     AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAE
                     AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSGRTATNFSLLKQAGD
                     VEENPG"
     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"
     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: 2020-10-29"
     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: #a6acb3"
                     /translation="GDA"
     CDS             2354..3085
                     /codon_start=1
                     /label=FMC63-28Z receptor protein
                     /note="color: #ffcc00"
                     /translation="DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVK
                     LLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKL
                     EITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIR
                     QPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAK
                     HYYYGGSYAMDYWGQGTSVTVS"
     CDS             2354..3076
                     /codon_start=1
                     /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     complement(3062..3114)
                     /label=CPR02
                     /note="color: black; sequence: 
                     ggtcgcggcgctggcgtcgtggtGGTACCggagacggtgactgaggttccttg; 
                     added: 2020-10-29"
     primer_bind     3062..3106
                     /label=CPF03
                     /note="color: black; sequence: 
                     caaggaacctcagtcaccgtctccGGTACCaccacgacgccagcg; added: 
                     2020-10-30"
     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..3298
                     /codon_start=1
                     /label=SEQ09 CD8 TM
                     /note="color: #99ccff"
                     /translation="TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD
                     IYIWAPLAGTCGVLLLSLVITLYC"
     CDS             3092..3226
                     /codon_start=1
                     /product="CD8 short hinge"
                     /label=CD8 hinge (CD276MG)
                     /note="nt sequence identical to human CD8a sequence"
                     /note="color: #ccffff"
                     /translation="TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD
                     "
     CDS             3092..3114
                     /codon_start=1
                     /label=CD8 Hinge
                     /note="color: #00ccff"
                     /translation="TTTPAPR"
     CDS             3227..3298
                     /codon_start=1
                     /label=CD8 TM
                     /note="color: #ccffff"
                     /translation="IYIWAPLAGTCGVLLLSLVITLYC"
     primer_bind     complement(3274..3321)
                     /label=CPR03
                     /note="color: black; sequence: 
                     tgcaggagcctgctcctcttactgcagtaaagggtgataaccagtgac; added: 
                     2020-10-30"
     primer_bind     complement(3274..3299)
                     /label=CPR03b
                     /note="color: black; sequence: 
                     agtttctttctgccccgtttgcagtaaagggtgataaccagtgac; added: 
                     2020-10-29"
     primer_bind     3277..3319
                     /label=CPF04
                     /note="color: black; sequence: 
                     actggttatcaccctttactgcagtaagaggagcaggctcctg; added: 
                     2020-10-29"
     primer_bind     3277..3299
                     /label=CPF04b
                     /note="color: black; sequence: 
                     actggttatcaccctttactgcaaacggggcagaaagaaactcc; added: 
                     2020-10-29"
     CDS             3299..3418
                     /codon_start=1
                     /label=CD28 CSD
                     /note="color: #33cccc"
                     /translation="SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS"
     primer_bind     3299..3319
                     /label=CPF04c
                     /note="color: black; sequence: 
                     ttattattttctgggtgaggagtaagaggagcaggctcctg; added: 
                     2020-10-29"
     primer_bind     3415..3440
                     /label=CPF04b
                     /note="color: black; sequence: 
                     actggttatcaccctttactgcaaacggggcagaaagaaactcc; added: 
                     2020-10-29"
     primer_bind     complement(3418..3438)
                     /label=CPR03b
                     /note="color: black; sequence: 
                     agtttctttctgccccgtttgcagtaaagggtgataaccagtgac; added: 
                     2020-10-29"
     CDS             3419..3544
                     /codon_start=1
                     /product="41BB CSD"
                     /label=41BB
                     /note="color: #0000ff"
                     /translation="KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL"
     primer_bind     3419..3440
                     /label=CPF04d
                     /note="color: black; sequence: 
                     ttattattttctgggtgaggaaacggggcagaaagaaactcc; added: 
                     2020-10-29"
     CDS             3545..3550
                     /codon_start=1
                     /label=Kpn2I
                     /note="color: #c0c0c0"
                     /translation="SG"
     CDS             3551..3892
                     /codon_start=1
                     /label=CD3z isoform 1 - no Esp3I
                     /note="color: #ff0000"
                     /translation="LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPE
                     MGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD
                     ALHMQALPPR"
     misc_feature    3667
                     /label=a->g
                     /note="a to g mutated to eliminate Esp3I site"
                     /note="color: #ffff00"
     primer_bind     complement(3883..3948)
                     /label=CPR04
                     /note="color: black; sequence: 
                     tcttccacgtcgcctgcttgctttaacagagagaagttcgtggccgtacgTCCGGAgcg
                     agggggc; added: 2020-10-29"
     CDS             3893..3898
                     /codon_start=1
                     /label=Kpn2I
                     /note="color: #c0c0c0"
                     /translation="SG"
     CDS             3899..3904
                     /codon_start=1
                     /label=BsiWI
                     /note="color: #c0c0c0"
                     /translation="RT"
     misc_feature    3904..4687
                     /label={2nd scFv-28TM-CD28-BB-CD3z CAR}
                     /label=nonstandard type:  ORF
                     /note="/application_notes="
                     /note="/official_designation={2nd scFv-28TM-CD28-BB-CD3z 
                     CAR}"
                     /note="/uuid=a7af8ce6d40a4636ba582c49665c9db3"
                     /note="color: #028a02; direction: RIGHT"
     CDS             3905..3961
                     /codon_start=1
                     /locus_tag="P2A"
                     /label=nonstandard type: Signal_peptide
                     /note="color: #ff9900"
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     complement(3929..3950)
                     /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"
     CDS             3959..4678
                     /codon_start=1
                     /label=EGFP after P2A
                     /note="color: #------"
                     /translation="PVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             3962..4678
                     /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: 3962 .. 3964 / #05fd14 / 1a
                        2: 3965 .. 4678 / #05fd14"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     protein_bind    complement(4688..4712)
                     /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    4726..5323
                     /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"
     CDS             complement(5201..5212)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /note="color: #cc99b2
                     Cleavage site after base 5200"
                     /translation="IEGR"
     LTR             5459..5639
                     /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"
     polyA_signal    5712..5846
                     /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"
     rep_origin      5872..6007
                     /label=SV40 ori
                     /note="SV40 origin of replication"
                     /note="color: #ffff00"
     rep_origin      5872..6007
                     /label=SV40 ori
                     /note="SV40 origin of replication"
                     /note="color: #ffff00"
     promoter        complement(6028..6046)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /note="color: #ffffff; direction: LEFT"
     primer_bind     complement(6056..6072)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: LEFT"
     rep_origin      6214..6669
                     /locus_tag="F1 ori"
                     /label=F1 ori
                     /note="color: #ffff00"
     promoter        6695..6799
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: RIGHT"
     CDS             6800..7660
                     /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: 6800 .. 6868 / #ccffcc / signal sequence
                        2: 6869 .. 7660 / #ccffcc
                     Cleavage site after base 6868"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      7831..8419
                     /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    8707..8728
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
                     /note="color: #31849b"
     protein_bind    8707..8728
                     /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        8743..8773
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
                     /note="This forward directional feature has 3 segments:
                        1: 8743 .. 8748 / #ffffff / -35
                        2: 8749 .. 8766 / #ffffff
                        3: 8767 .. 8773 / #ffffff / -10"
     protein_bind    8781..8797
                     /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     8805..8821
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: RIGHT"
     promoter        8842..8860
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
                     /note="color: #ffffff; direction: RIGHT"
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 caccacgacg ccagcgccgc gaccaccaac
     3121 accggcgccc accatcgcgt cgcagcccct gtccctgcgc ccagaggcgt gccggccagc
     3181 ggcggggggc gcagtgcaca cgagggggct ggacttcgcc tgtgatatct acatctgggc
     3241 gcccttggcc gggacttgtg gggtccttct cctgtcactg gttatcaccc tttactgcag
     3301 taagaggagc aggctcctgc acagtgacta catgaacatg actcccaggc ggcccggacc
     3361 cacccgcaag cattaccagc cctatgcccc accacgcgac ttcgcagcct atcgctccaa
     3421 acggggcaga aagaaactcc tgtatatatt caaacaacca tttatgagac cagtacaaac
     3481 tactcaagag gaagatggct gtagctgccg atttccagaa gaagaagaag gaggatgtga
     3541 actgtccgga ctgagagtga agttcagcag gagcgcagac gcccccgcgt accagcaggg
     3601 ccagaaccag ctctataacg agctcaatct aggacgaaga gaggagtacg atgttttgga
     3661 caagaggcgt ggccgggacc ctgagatggg gggaaagccg cagagaagga agaaccctca
     3721 ggaaggcctg tacaatgaac tgcagaaaga taagatggcg gaggcctaca gtgagattgg
     3781 gatgaaaggc gagcgccgga ggggcaaggg gcacgatggc ctttaccagg gtctcagtac
     3841 agccaccaag gacacctacg acgcccttca catgcaggcc ctgccccctc gctccggacg
     3901 tacggccacg aacttctctc tgttaaagca agcaggcgac gtggaagaaa accccggtcc
     3961 cgtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     4021 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
     4081 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     4141 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
     4201 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
     4261 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     4321 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     4381 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
     4441 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     4501 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     4561 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     4621 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaagg
     4681 gccccgtacc cagctttctt gtacaaagtg gtgataatcg aattccgata atcaacctct
     4741 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
     4801 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
     4861 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt
     4921 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat
     4981 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc
     5041 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
     5101 caattccgtg gtgttgtcgg ggaagctgac gtcctttcca tggctgctcg cctgtgttgc
     5161 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga
     5221 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc
     5281 tcagacgagt cggatctccc tttgggccgc ctccccgcat cgggaattcc cgcggttcgc
     5341 tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg
     5401 gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg cttgtactgg
     5461 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
     5521 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
     5581 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
     5641 tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat gaatatcaga
     5701 gagtgagagg aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     5761 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     5821 caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc catcccgccc
     5881 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     5941 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     6001 ggaggcctag ggacgtaccc aattcgccct atagtgagtc gtattacgcg cgctcactgg
     6061 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     6121 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     6181 cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     6241 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     6301 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     6361 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     6421 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     6481 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     6541 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     6601 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     6661 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     6721 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     6781 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     6841 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     6901 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     6961 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     7021 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     7081 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     7141 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     7201 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     7261 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     7321 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     7381 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     7441 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     7501 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     7561 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     7621 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     7681 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     7741 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     7801 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     7861 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     7921 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     7981 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     8041 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     8101 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     8161 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     8221 gctatgagaa agcgccacgc ttcccgaaga gagaaaggcg gacaggtatc cggtaagcgg
     8281 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     8341 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     8401 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     8461 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     8521 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     8581 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
     8641 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
     8701 cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc
     8761 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga
     8821 ccatgattac gccaagcgcg caattaaccc tcactaaagg gaacaaaagc tggagctgca
     8881 agctt
//
