LOCUS       pCAG-CBE4max-SpR       11222 bp ds-DNA     circular SYN 05-JAN-2021
DEFINITION  CAG promoter expression plasmid for human codon optimized BE4max
            C-to-T base editor with
            SpRY(D10A/A61R/L1111R/D1135L/S1136W/G1218K/E1219Q/N1317R/A1322R/R13
            3 3P/R1335Q/T1337R) and P2A-EGFP.
ACCESSION
VERSION     .
KEYWORDS    pCAG-CBE4max-SpRY-P2A-EGFP (RTW5133).
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 11195)
  AUTHORS   Kleinstiver
  TITLE     Kleinstiver lab CRISPR plasmids
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 11195)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Mar 20, 2020 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     enhancer        37..416
                     /note="human cytomegalovirus immediate early enhancer"
                     /label="CMV enhancer"
     promoter        418..693
                     /label="chicken beta-actin promoter"
     intron          694..1710
                     /note="chimera between introns from chicken beta-actin and
                      rabbit beta-globin"
                     /label="chimeric intron"
     primer_bind     1634..1656
                     /note="Chimeric intron in CAG promoter, forward primer"
                     /label="pCAGGS-5"
     primer_bind     1718..1737
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
                     /label="pCAG-F"
     CDS             1845..1865
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian
                     virus 40) large T antigen"
                     /translation="PKKKRKV"
                     /label="SV40 NLS"
     CDS             1866..2549
                     /codon_start=1
                     /product="cytidine deaminase (C to U editing enzyme) from
                     rat"
                     /note="can use ssDNA as a substrate (Komor et al., 2016)"
                     /translation="SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
                     GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEF
                     LSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSP
                     SNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLP
                     PHILWATGLK"
                     /label="APOBEC-1"
     misc_feature    complement(2604..2623)
                     /created_by="primer3"
                     /%GC="55.0"
                     /Tm="60.0"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="CTGCTCTCTGGTGTTGCTGA"
                     /annotation_group="20664841607202696716ee27-44a0-4de0-ac63
                     -badaafc73400urn:sequence:genbank:."
                     /label="2,623 R"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(2604..2663)
                     /created_by="User"
                     /Sequence="GATGGAGTATTTCTTCTCGCTGCCGCCGCTGCTGCCGCCACTGCTCT
                     CTGGTGTTGCTGA"
                     /modified_by="User"
                     /label
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    2632..2691
                     /Sequence="GCAGCGGCGGCAGCGAGAAGAAATACTCCATCGGCCTGGCCATCGGA
                     ACCAACAGCGTGG"
                     /created_by="User"
                     /label
                     /note="Geneious type: primer_bind_reverse"
     source          <2646..>5975
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     ORF             <2646..>5975
                     /created_by="User"
                     /modified_by="User"
                     /label="Sc++"
     primer_bind     2646..2691
                     /Sequence="GAGAAGAAATACTCCATCGGCCTGGCCATCGGAACCAACAGCGTGG"
                     /created_by="User"
                     /label
     misc_feature    2648..5975
                     /Original_Bases="CAAGAAGTACAGCATCGGCCTGGCCATCGGCACCAACTCTG
                     TGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCT
                     GGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGAC
                     AGCGGCGAAACAGCCGAGAGAACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCA
                     GACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGT
                     GGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAG
                     CACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGT
                     ACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCT
                     GCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATC
                     GAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGC
                     AGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAA
                     GGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAG
                     CTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCC
                     TGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAG
                     CAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTAC
                     GCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCC
                     TGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATA
                     CGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCT
                     GAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTG
                     ACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGAT
                     GGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAG
                     CGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCA
                     TTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGA
                     GAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGC
                     AGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGG
                     AAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGA
                     TAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTC
                     ACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCG
                     CCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCG
                     GAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGAC
                     TCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACG
                     ATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACAT
                     TCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAA
                     CGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGC
                     GGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAA
                     GCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAAC
                     TTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCC
                     AGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCC
                     CGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTG
                     ATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCA
                     CCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAA
                     AGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAAC
                     GAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAAC
                     TGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCT
                     GAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAG
                     AGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGC
                     TGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAG
                     AGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACC
                     CGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACG
                     ACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGT
                     GTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCAC
                     CACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACC
                     CTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGAT
                     GATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGC
                     AACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGC
                     GGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGA
                     TTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACC
                     GAGGTGCAGACA"
                     /label="CAAGAAGTACAGCATCGGCCTGGCCATCGGCACCAACTCTGTGGGCTGGG
                     CCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACAC
                     CGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAA
                     ACAGCCGAGAGAACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGA
                     ACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAG
                     CTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGG
                     CACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCA
                     TCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGAT
                     CTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGAC
                     CTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACA
                     ACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCT
                     GTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGC
                     GAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCA
                     ACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACAC
                     CTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTG
                     TTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGA
                     ACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCA
                     CCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTAC
                     AAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAG
                     CCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCAC
                     CGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTC
                     GACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGC
                     GGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCT
                     GACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCC
                     TGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGG
                     ACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCT
                     GCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTAT
                     AACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGA
                     GCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGAC
                     CGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAA
                     ATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGA
                     AAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGA
                     TATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAA
                     ACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACA
                     CCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGG
                     CAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAG
                     CTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCG
                     GCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAA
                     GAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGG
                     CACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGG
                     GACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGG
                     CAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTG
                     TACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCA
                     ACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGA
                     CTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAAC
                     GTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACG
                     CCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCT
                     GAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATC
                     ACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATG
                     ACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTT
                     CCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCAC
                     GACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGG
                     AAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAA
                     GAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATG
                     AACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGA
                     TCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCAC
                     CGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAG
                     ACA"
                     /note="Geneious type: Editing History Replacement"
     primer_bind     2673..2691
                     /created_by="primer3"
                     /%GC="57.9"
                     /Tm="60.7"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="ATCGGAACCAACAGCGTGG"
                     /annotation_group="460569505809987feb6eefe-33ec-4892-b980-
                     1e205872f457urn:sequence:genbank:."
                     /label="3,052 F"
     primer_bind     complement(2849..2863)
                     /Sequence="CTGGTGTACCGTCTT"
                     /Hairpin_Tm="39.4"
                     /Tm="48.1"
                     /Self_Dimer_Tm="None"
                     /%GC="53.3"
                     /created_by="primer3"
                     /label
     Junction        3312
                     /Reads_using=3
                     /Reads_supporting_discovery=3
                     /Rearrangement_Distance=1317
                     /Junction_Type="Rearrangement"
                     /Junction_Source="3386+"
                     /Junction_Destination="4704+"
                     /label="junction 1"
     misc_feature    3735..3764
                     /created_by="User"
                     /modified_by="User"
                     /label="Loop from S. anginosus"
                     /note="Geneious type: topological domain"
     Junction        4630
                     /Reads_using=3
                     /Reads_supporting_discovery=3
                     /Rearrangement_Distance=1317
                     /Junction_Type="Rearrangement"
                     /Junction_Source="4704-"
                     /Junction_Destination="3386-"
                     /label="junction 1"
     Junction        order(4742,4746)
                     /Reads_using=5
                     /Reads_supporting_discovery=2
                     /Deletion_Size=3
                     /Junction_Type="Deletion"
                     /Junction_Source="4813+"
                     /Junction_Destination="4817+"
                     /label="junction 1"
     Junction        order(4742,4746)
                     /Reads_using=2
                     /Reads_supporting_discovery=2
                     /Deletion_Size=3
                     /Junction_Type="Deletion"
                     /Junction_Source="4813+"
                     /Junction_Destination="4817+"
                     /label="junction 1"
     primer_bind     5055..5069
                     /Sequence="CTGAAGGAAAACCCC"
                     /Hairpin_Tm="34.7"
                     /Tm="46.8"
                     /Self_Dimer_Tm="None"
                     /%GC="53.3"
                     /created_by="primer3"
                     /label
     misc_feature    complement(5952..5971)
                     /created_by="primer3"
                     /%GC="55.0"
                     /Tm="60.0"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="TGCACCTCGGTCTTCTTCAC"
                     /annotation_group="1258366549447096ad5a485c-8fc5-4d29-bec7
                     -a46e95259297urn:sequence:genbank:."
                     /label="5,470 R"
                     /note="Geneious type: primer_bind_reverse"
     ORF             <5976..6773
                     /created_by="User"
                     /label
     gene            6000..6773
                     /created_by="User"
                     /modified_by="User"
                     /label="SpRY PID"
     CDS             6804..7052
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus
                      subtilis bacteriophage (Mol et al., 1995)"
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHT
                     AYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
                     /label="UGI"
     CDS             7083..7331
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus
                      subtilis bacteriophage (Mol et al., 1995)"
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHT
                     AYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
                     /label="UGI"
     CDS             7374..7394
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian
                     virus 40) large T antigen"
                     /translation="PKKKRKV"
                     /label="SV40 NLS"
     CDS             7413..7469
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /note="Eukaryotic ribosomes fail to insert a peptide bond
                     between the Gly and Pro residues, yielding
                     separatepolypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
                     /label="P2A"
     CDS             7470..8189
                     /codon_start=1
                     /product="enhanced GFP"
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT
                     LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFK
                     DDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKN
                     GIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDH
                     MVLLEFVTAAGITLGMDELYK"
                     /label="EGFP"
     primer_bind     complement(7515..7536)
                     /note="EGFP, reverse primer"
                     /label="EGFP-N"
     primer_bind     complement(7776..7795)
                     /note="For distinguishing EGFP variants, reverse primer"
                     /label="EXFP-R"
     primer_bind     8123..8144
                     /note="EGFP, forward primer"
                     /label="EGFP-C"
     primer_bind     complement(8276..8295)
                     /note="Rabbit beta-globin polyA region, reverse primer"
                     /label="Bglob-pA-R"
     polyA_site      8341..8396
                     /note="rabbit beta-globin polyadenylation signal"
                     /label="-beta-globin poly(A) signal"
     primer_bind     complement(8395..8414)
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
                     /label="rbglobpA-R"
     primer_bind     complement(8757..8773)
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /label="M13 rev"
     primer_bind     complement(8757..8773)
                     /note="In lacZ gene. Also called M13-rev"
                     /label="M13 Reverse"
     primer_bind     complement(8770..8792)
                     /note="In lacZ gene"
                     /label="M13/pUC Reverse"
     protein_bind    8781..8797
                     /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)."
                     /label="lac operator"
     promoter        complement(8805..8835)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    8850..8871
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                      of cAMP."
                     /label="CAP binding site"
     promoter        8930..9125
                     /note="SV40 early promoter"
                     /label="SV40 promoter"
     rep_origin      8976..9111
                     /note="SV40 origin of replication"
                     /label="SV40 ori"
     primer_bind     9038..9057
                     /note="SV40 promoter/origin, forward primer"
                     /label="SV40pro-F"
     polyA_signal    9131..9265
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     primer_bind     complement(9168..9187)
                     /note="SV40 polyA, reverse primer"
                     /label="SV40pA-R"
     primer_bind     9222..9241
                     /note="SV40 polyA terminator, reverse primer"
                     /label="EBV-rev"
     primer_bind     complement(9333..9350)
                     /note="L4440 vector, forward primer"
                     /label="L4440"
     rep_origin      complement(9504..10092)
                     /direction="LEFT"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /label="ori"
     primer_bind     complement(9584..9603)
                     /note="pBR322 origin, forward primer"
                     /label="pBR322ori-F"
     CDS             complement(10263..11123)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="confers resistance to ampicillin, carbenicillin,
                     and related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW"
                     /label="AmpR"
     primer_bind     10886..10905
                     /note="Ampicillin resistance gene, reverse primer"
                     /label="Amp-R"
     promoter        complement(11124..6)
                     /gene="bla"
                     /label="AmpR promoter"
ORIGIN      
        1 tccgcgcaca tttccccgaa aagtgccacc tgggtcgaca ttgattattg actagttatt
       61 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
      121 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
      181 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
      241 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
      301 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
      361 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg
      421 aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt
      481 tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggcgc
      541 gcgccaggcg gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg
      601 gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg
      661 cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc
      721 gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc
      781 ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagcg cttggtttaa
      841 tgacggcttg tttcttttct gtggctgcgt gaaagccttg aggggctccg ggagggccct
      901 ttgtgcgggg ggagcggctc ggggggtgcg tgcgtgtgtg tgtgcgtggg gagcgccgcg
      961 tgcggctccg cgctgcccgg cggctgtgag cgctgcgggc gcggcgcggg gctttgtgcg
     1021 ctccgcagtg tgcgcgaggg gagcgcggcc gggggcggtg ccccgcggtg cggggggggc
     1081 tgcgagggga acaaaggctg cgtgcggggt gtgtgcgtgg gggggtgagc agggggtgtg
     1141 ggcgcgtcgg tcgggctgca accccccctg cacccccctc cccgagttgc tgagcacggc
     1201 ccggcttcgg gtgcggggct ccgtacgggg cgtggcgcgg ggctcgccgt gccgggcggg
     1261 gggtggcggc aggtgggggt gccgggcggg gcggggccgc ctcgggccgg ggagggctcg
     1321 ggggaggggc gcggcggccc ccggagcgcc ggcggctgtc gaggcgcggc gagccgcagc
     1381 cattgccttt tatggtaatc gtgcgagagg gcgcagggac ttcctttgtc ccaaatctgt
     1441 gcggagccga aatctgggag gcgccgccgc accccctcta gcgggcgcgg ggcgaagcgg
     1501 tgcggcgccg gcaggaagga aatgggcggg gagggccttc gtgcgtcgcc gcgccgccgt
     1561 ccccttctcc ctctccagcc tcggggctgt ccgcgggggg acggctgcct tcggggggga
     1621 cggggcaggg cggggttcgg cttctggcgt gtgaccggcg gctctagagc ctctgctaac
     1681 catgttcatg ccttcttctt tttcctacag ctcctgggca acgtgctggt tattgtgctg
     1741 tctcatcatt ttggcaaaga attctgcagt cgacggtacc gcgggcccgg gatccaccgg
     1801 tcgccaccat gaaacggaca gccgacggaa gcgagttcga gtcaccaaag aagaagcgga
     1861 aagtctcctc agagactggg cctgtcgccg tcgatccaac cctgcgccgc cggattgaac
     1921 ctcacgagtt tgaagtgttc tttgaccccc gggagctgag aaaggagaca tgcctgctgt
     1981 acgagatcaa ctggggaggc aggcactcca tctggaggca cacctctcag aacacaaata
     2041 agcacgtgga ggtgaacttc atcgagaagt ttaccacaga gcggtacttc tgccccaata
     2101 ccagatgtag catcacatgg tttctgagct ggtccccttg cggagagtgt agcagggcca
     2161 tcaccgagtt cctgtccaga tatccacacg tgacactgtt tatctacatc gccaggctgt
     2221 atcaccacgc agacccaagg aataggcagg gcctgcgcga tctgatcagc tccggcgtga
     2281 ccatccagat catgacagag caggagtccg gctactgctg gcggaacttc gtgaattatt
     2341 ctcctagcaa cgaggcccac tggcctaggt acccacacct gtgggtgcgc ctgtacgtgc
     2401 tggagctgta ttgcatcatc ctgggcctgc ccccttgtct gaatatcctg cggagaaagc
     2461 agccccagct gaccttcttt acaatcgccc tgcagtcttg tcactatcag aggctgccac
     2521 cccacatcct gtgggccaca ggcctgaagt ctggaggatc tagcggagga tcctctggca
     2581 gcgagacacc aggaacaagc gagtcagcaa caccagagag cagtggcggc agcagcggcg
     2641 gcagcgagaa gaaatactcc atcggcctgg ccatcggaac caacagcgtg ggctgggccg
     2701 tgatcaccga cgactataag gtgcccagca agaagttcaa agtgctgggc aacacaaaca
     2761 gaaagagcat caagaagaac ctgatgggcg ccctgctgtt cgactccgga gagaccgccg
     2821 aagcaacccg gctgaagcgc accgccagaa gacggtacac cagaagaaaa aacaggatca
     2881 gatacctgca ggagatcttc gccaacgaga tggccaaact ggacgatagc tttttccagc
     2941 ggctggaaga atccttcctg gtggaggagg acaagaaaaa cgagagacac cctatctttg
     3001 ggaacctggc cgacgaggtg gcctaccaca gaaactaccc caccatctac cacctgagaa
     3061 agaagctggc cgactccccc gaaaaggccg acctgagact gatctacctc gccctggccc
     3121 acatcatcaa gtttcggggc cacttcctga ttgaaggcaa gctgaacgcc gagaactccg
     3181 acgtggccaa gctgttctac cagctgatcc agacctacaa tcagctgttt gaggaaagcc
     3241 ctctggacga aatcgaagtg gacgccaaag ggatcctgtc cgcccggctg agcaagtcta
     3301 agcggctgga gaagctgatt gccgtcttcc ccaacgagaa gaaaaacggc ctgttcggca
     3361 acattatcgc cctggccctg ggcctgaccc ccaactttaa gagcaatttc gatctgaccg
     3421 aggatgccaa gctgcagctg tccaaggata cctacgacga cgatctggac gagctgctgg
     3481 gacagatcgg agaccagtac gccgacctgt tcagcgccgc caaaaacctg tccgatgcca
     3541 tcctgctgag cgacatcctg cggagcaaca gcgaagtcac caaagcgccc ctgagcgcca
     3601 gcatggtgaa gcggtacgac gagcaccacc aggacctggc gctgctgaag acactggtga
     3661 gacagcagtt ccccgagaag tacgccgaga tcttcaagga cgataccaaa aatggctacg
     3721 ctggctacgt gggagctgat aagaagctgc ggaagcggtc cggcaagctg gccaccgagg
     3781 aggagtttta caagttcatc aagccgatcc tggaaaagat ggatggagcc gaagagctgc
     3841 tggccaagct gaaccgggac gacctgctga gaaagcaacg gaccttcgac aacggatcca
     3901 tccctcacca gatccacctg aaagagctgc acgctatcct gagacggcaa gaagaatttt
     3961 accccttcct gaaggagaac cgggagaaga tcgaaaagat cctgaccttc agaatcccct
     4021 actacgtggg gccactggcc cggggcaata gcagattcgc ctggctgacc cggaagtctg
     4081 aagaggccat caccccctgg aattttgagg aggtggtgga caaaggcgcc agcgcccagt
     4141 ccttcatcga gcggatgacc aacttcgatg agcagctgcc aaacaagaag gtcctgccca
     4201 agcactctct gctgtacgaa tattttaccg tgtacaacga actgaccaaa gtgaagtacg
     4261 tgactgagag aatgcggaag cctgaattcc tgtccggaga gcagaagaaa gccatcgtgg
     4321 acctgctgtt caagaccaac agaaaagtga ccgtgaaaca gctgaaggag gactacttca
     4381 agaagatcga gtgcttcgac agcgtggaga ttatcggagt ggaagacaga ttcaacgcca
     4441 gcctgggcac ctaccacgat ctgctgaaga tcatcaagga taaggacttc ctggacaacg
     4501 aggagaacga agacatcctg gaggacatcg tgctgacact gaccctgttc gaggaccggg
     4561 aaatgatcga agaacggctc aaaacatacg ctcacctgtt cgatgacaag gtgatgaagc
     4621 agctgaagag aagacactac accggctggg gccggctgtc ccggaagatg atcaacggaa
     4681 tccgggacaa gcagtccggc aagacaatcc tggacttcct gaagtccgac ggcttcagca
     4741 acagaaactt catgcagctg atccacgacg acagcctgac ctttaaagag gagatcgaga
     4801 aagcccaggt gtccggccaa ggagactctc tgcacgagca gatcgccgac ctggccggca
     4861 gccccgccat caaaaaaggc attctgcaga ccgtgaagat cgtggatgag ctggtgaaag
     4921 tgatgggcca caagcctgag aacatcgtga tcgagatggc cagagagaac cagaccacca
     4981 ccaaaggact gcagcagtcc agggagcgga aaaagcggat cgaggaaggc atcaaggagc
     5041 tggaaagcca gattctgaag gaaaaccccg tggaaaacac ccagctgcag aacgaaaagc
     5101 tgtacctgta ctacctgcag aatggcagag atatgtacgt ggatcaggag ctggatatca
     5161 atcggctgtc cgactacgat gtggaccaca tcgtgcccca gagcttcatc aaggacgact
     5221 ccatcgataa caaagtgctg acccggtctg tggagaacag aggcaagagc gacaacgtgc
     5281 ccagcgaaga agtggtgaag aagatgaaga actactggcg gcagctgctg aacgccaagc
     5341 tgatcaccca gagaaagttc gacaacctga ccaaggccga gcggggcggc ctgagcgaag
     5401 ccgataaggc tggcttcatc aagcggcagc tggtggaaac caggcagatt acaaagcacg
     5461 tggcccggat cctggacagc agaatgaaca ccaagagaga caagaacgac aagcccatcc
     5521 gggaggtgaa ggtgatcacc ctgaagagca aactggtgag cgacttcaga aaagacttcc
     5581 agctgtacaa agtgcgggac atcaacaact accaccacgc ccacgacgcc tacctgaacg
     5641 ccgtggtggg caccgctctg atcaagaagt accccaagct ggagagcgag ttcgtgtacg
     5701 gcgattataa ggtgtacgat gtgcggaaaa tgatcgccaa gagcgagcag gagatcggca
     5761 aggccaccgc caaacggttc ttctacagca acatcatgaa tttcttcaag accgaggtga
     5821 agctggccaa cggcgagatc agaaagagac ctctgattga aaccaacggc gagaccggcg
     5881 aagtggtgtg gaacaaggag aaggacttcg ccaccgtgcg gaaggtgctg gccatgcccc
     5941 aggtgaacat cgtgaagaag accgaggtgc agactggcgg cttcagcaaa gagtctatca
     6001 gacccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac cctaagaagt
     6061 acggcggctt cctgtggccc accgtggcct attctgtgct ggtggtggcc aaagtggaaa
     6121 agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc atcatggaaa
     6181 gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac aaagaagtga
     6241 aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa aacggccgga
     6301 agagaatgct ggcctctgcc aagcagctgc agaagggaaa cgaactggcc ctgccctcca
     6361 aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc tcccccgagg
     6421 ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac gagatcatcg
     6481 agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg gacaaagtgc
     6541 tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag aatatcatcc
     6601 acctgtttac cctgaccaga ctgggagccc ctagagcctt caagtacttt gacaccacca
     6661 tcgaccccaa gcagtacaga agcaccaaag aggtgctgga cgccaccctg atccaccaga
     6721 gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggt gacagcggcg
     6781 ggagcggcgg gagcgggggg agcactaatc tgagcgacat cattgagaag gagactggga
     6841 aacagctggt cattcaggag tccatcctga tgctgcctga ggaggtggag gaagtgatcg
     6901 gcaacaagcc agagtctgac atcctggtgc acaccgccta cgacgagtcc acagatgaga
     6961 atgtgatgct gctgacctct gacgcccccg agtataagcc ttgggccctg gtcatccagg
     7021 attctaacgg cgagaataag atcaagatgc tgagcggagg atccggagga tctggaggca
     7081 gcaccaacct gtctgacatc atcgagaagg agacaggcaa gcagctggtc atccaggaga
     7141 gcatcctgat gctgcccgaa gaagtcgaag aagtgatcgg aaacaagcct gagagcgata
     7201 tcctggtcca taccgcctac gacgagagta ccgacgaaaa tgtgatgctg ctgacatccg
     7261 acgccccaga gtataagccc tgggctctgg tcatccagga ttccaacgga gagaacaaaa
     7321 tcaaaatgct gtctggcggc tcaaaaagaa ccgccgacgg cagcgaattc gagcccaaga
     7381 agaagaggaa agtcggaggc ggtggaagcg gcgcaacaaa cttctctctg ctgaaacaag
     7441 ccggagatgt cgaagagaat cctggaccga tggtgagcaa gggcgaggag ctgttcaccg
     7501 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     7561 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     7621 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     7681 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     7741 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     7801 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     7861 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     7921 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     7981 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     8041 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     8101 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     8161 ctctcggcat ggacgagctg tacaagtgaa gcggccgcac tcctcaggtg caggctgcct
     8221 atcagaaggt ggtggctggt gtggccaatg ccctggctca caaataccac tgagatcttt
     8281 ttccctctgc caaaaattat ggggacatca tgaagcccct tgagcatctg acttctggct
     8341 aataaaggaa atttattttc attgcaatag tgtgttggaa ttttttgtgt ctctcactcg
     8401 gaaggacata tgggagggca aatcatttaa aacatcagaa tgagtatttg gtttagagtt
     8461 tggcaacata tgcccatatg ctggctgcca tgaacaaagg ttggctataa agaggtcatc
     8521 agtatatgaa acagccccct gctgtccatt ccttattcca tagaaaagcc ttgacttgag
     8581 gttagatttt ttttatattt tgttttgtgt tatttttttc tttaacatcc ctaaaatttt
     8641 ccttacatgt tttactagcc agatttttcc tcctctcctg actactccca gtcatagctg
     8701 tccctcttct cttatggaga tccctcgacc tgcagcccaa gcttggcgta atcatggtca
     8761 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     8821 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     8881 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agcggatccg catctcaatt
     8941 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     9001 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     9061 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     9121 gcaaaaagct aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     9181 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     9241 caatgtatct tatcatgtct ggatccgctg cattaatgaa tcggccaacg cgcggggaga
     9301 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     9361 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     9421 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     9481 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     9541 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     9601 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     9661 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     9721 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     9781 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     9841 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     9901 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     9961 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
    10021 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
    10081 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
    10141 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
    10201 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
    10261 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
    10321 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
    10381 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
    10441 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
    10501 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
    10561 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
    10621 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
    10681 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
    10741 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
    10801 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
    10861 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
    10921 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
    10981 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
    11041 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
    11101 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
    11161 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
    11221 gt
//
