LOCUS       pCMV-T7-SpRYc-P2        8478 bp ds-DNA     circular SYN 19-AUG-2021
DEFINITION  PAM-Free nuclease with EGFP tag.
ACCESSION
VERSION     .
KEYWORDS    pCMV-T7-SpRY-P2A-EGFP (RTW4830).
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 8451)
  AUTHORS   Kleinstiver
  TITLE     Kleinstiver lab CRISPR plasmids
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 8451)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Mar 20, 2020 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     CDS             complement(250..1110)
                     /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"
     promoter        complement(1111..1215)
                     /gene="bla"
                     /label="AmpR promoter"
     enhancer        1485..1864
                     /note="human cytomegalovirus immediate early enhancer"
                     /label="CMV enhancer"
     promoter        1865..2068
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
                     /label="CMV promoter"
     primer_bind     2036..2055
                     /created_by="primer3"
                     /%GC="60.0"
                     /Tm="60.7"
                     /Hairpin_Tm="35.4"
                     /Self_Dimer_Tm="None"
                     /Sequence="CGTGTACGGTGGGAGGTCTA"
                     /annotation_group="10536599603609656774a780-afec-4924-a671
                     -97a857f5d357urn:sequence:genbank:."
                     /label="2,036 F"
     promoter        2110..2128
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /label="T7 promoter"
     regulatory      2136..>2141
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation
                      of translation (Kozak, 1987)"
     ORF             2142..>5501
                     /created_by="User"
                     /modified_by="User"
                     /label="Sc++ N-Term"
     misc_feature    2147..5474
                     /Original_Bases="CAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTG
                     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="CAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGG
                     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     2556..2575
                     /created_by="primer3"
                     /%GC="55.0"
                     /Tm="60.0"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="AGAAAGAAGCTGGCCGACTC"
                     /annotation_group="105367157284358281f6fec6-30ac-4484-bf1f
                     -7f8dca4db517urn:sequence:genbank:."
                     /label="2,556 F"
     Junction        2811
                     /Reads_using=3
                     /Reads_supporting_discovery=3
                     /Rearrangement_Distance=1317
                     /Junction_Type="Rearrangement"
                     /Junction_Source="3386+"
                     /Junction_Destination="4704+"
                     /label="junction 1"
     primer_bind     3017..3036
                     /created_by="primer3"
                     /%GC="55.0"
                     /Tm="60.1"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="CGCCAAAAACCTGTCCGATG"
                     /annotation_group="1053680054584036e94937fd-05ad-41d5-af6a
                     -c73ac92a6d50urn:sequence:genbank:."
                     /label="3,017 F"
     misc_feature    3234..3263
                     /created_by="User"
                     /modified_by="User"
                     /label="Loop from S. anginosus"
                     /note="Geneious type: topological domain"
     primer_bind     3606..3625
                     /created_by="primer3"
                     /%GC="60.0"
                     /Tm="60.0"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="GAGGAGGTGGTGGACAAAGG"
                     /annotation_group="1053687519077104e744cfe8-6238-400c-9523
                     -8935763322a2urn:sequence:genbank:."
                     /label="3,606 F"
     Junction        4129
                     /Reads_using=3
                     /Reads_supporting_discovery=3
                     /Rearrangement_Distance=1317
                     /Junction_Type="Rearrangement"
                     /Junction_Source="4704-"
                     /Junction_Destination="3386-"
                     /label="junction 1"
     primer_bind     4192..4211
                     /created_by="primer3"
                     /%GC="55.0"
                     /Tm="60.0"
                     /Hairpin_Tm="39.3"
                     /Self_Dimer_Tm="8.0"
                     /Sequence="AGTCCGGCAAGACAATCCTG"
                     /annotation_group="1053695792380512fbab41dc-ccaa-4b47-bd70
                     -cea008eb2ef8urn:sequence:genbank:."
                     /label="4,192 F"
     Junction        order(4241,4245)
                     /Reads_using=5
                     /Reads_supporting_discovery=2
                     /Deletion_Size=3
                     /Junction_Type="Deletion"
                     /Junction_Source="4813+"
                     /Junction_Destination="4817+"
                     /label="junction 1"
     Junction        order(4241,4245)
                     /Reads_using=2
                     /Reads_supporting_discovery=2
                     /Deletion_Size=3
                     /Junction_Type="Deletion"
                     /Junction_Source="4813+"
                     /Junction_Destination="4817+"
                     /label="junction 1"
     primer_bind     4753..4772
                     /created_by="primer3"
                     /%GC="55.0"
                     /Tm="60.0"
                     /Hairpin_Tm="43.4"
                     /Self_Dimer_Tm="None"
                     /Sequence="AGAACAGAGGCAAGAGCGAC"
                     /annotation_group="10537026363397291624370e-ee25-46f6-ac3c
                     -c00c8351580burn:sequence:genbank:."
                     /label="4,753 F"
     primer_bind     5220..5239
                     /created_by="primer3"
                     /%GC="50.0"
                     /Tm="59.8"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="GTGCGGAAAATGATCGCCAA"
                     /annotation_group="1053712059254913eaf6b562-3250-4860-993a
                     -abe6f5f67697urn:sequence:genbank:."
                     /label="5,220 F"
     gene            <5502..6272
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes
                     Cas9 (Zetsche et al., 2015)"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIK
                     KNLIGALLFDSGETAERTRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE
                     ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH
                     MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSK
                     SRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLD
                     NLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTL
                     LKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVK
                     LNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY
                     YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVL
                     PKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKE
                     DYFKKIE"
                     /modified_by="User"
                     /label="SpRY PID"
     primer_bind     5769..5788
                     /created_by="primer3"
                     /%GC="55.0"
                     /Tm="60.0"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="16.4"
                     /Sequence="TACTCCCTGTTCGAGCTGGA"
                     /annotation_group="105372057786830869a990d5-06a4-4119-a184
                     -d8b3ea020a03urn:sequence:genbank:."
                     /label="5,769 F"
     primer_bind     6164..6183
                     /created_by="primer3"
                     /%GC="55.0"
                     /Tm="60.0"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="3.2"
                     /Sequence="CCCCAAGCAGTACAGAAGCA"
                     /annotation_group="10537669705504728779639c-1200-4ae3-adec
                     -840e22941203urn:sequence:genbank:."
                     /label="6,164 F"
     CDS             6327..6347
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian
                     virus 40) large T antigen"
                     /translation="PKKKRKV"
                     /label="SV40 NLS"
     CDS             6360..6425
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by
                     an enterokinase cleavage site"
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
                     /label="3xFLAG"
     CDS             6444..6500
                     /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             6501..7220
                     /codon_start=1
                     /product="enhanced GFP"
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT
                     LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFK
                     DDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKN
                     GIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDH
                     MVLLEFVTAAGITLGMDELYK"
                     /label="EGFP"
     primer_bind     6566..6585
                     /created_by="primer3"
                     /%GC="50.0"
                     /Tm="59.1"
                     /Hairpin_Tm="35.4"
                     /Self_Dimer_Tm="None"
                     /Sequence="CGTAAACGGCCACAAGTTCA"
                     /annotation_group="105377576712178292c98891-2e4e-4196-a930
                     -9f8938732c1burn:sequence:genbank:."
                     /label="6,566 F"
     primer_bind     7089..7108
                     /created_by="primer3"
                     /%GC="60.0"
                     /Tm="59.8"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="CCCGACAACCACTACCTGAG"
                     /annotation_group="10537841430071744c42ac1a-c0bd-4a24-9239
                     -96ff32ed9342urn:sequence:genbank:."
                     /label="7,089 F"
     CDS             7226..7243
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /translation="HHHHHH"
                     /label="6xHis"
     polyA_signal    7272..7496
                     /note="bovine growth hormone polyadenylation signal"
                     /label="bGH poly(A) signal"
     protein_bind    7591..7607
                     /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(7615..7645)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    7660..7681
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                      of cAMP."
                     /label="CAP binding site"
     misc_feature    complement(7848..7867)
                     /created_by="primer3"
                     /%GC="55.0"
                     /Tm="60.0"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="TACCGCCTTTGAGTGAGCTG"
                     /annotation_group="1053809094922990952778ec-2626-4238-a984
                     -5471fb747dedurn:sequence:genbank:."
                     /label="7,867 R"
                     /note="Geneious type: primer_bind_reverse"
     rep_origin      complement(7969..79)
                     /direction="LEFT"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /label="ori"
ORIGIN      
        1 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
       61 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
      121 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
      181 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
      241 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
      301 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
      361 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
      421 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
      481 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
      541 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
      601 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
      661 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
      721 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
      781 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
      841 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
      901 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
      961 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     1021 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     1081 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     1141 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     1201 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga
     1261 tcgatctccc gatcccctag ggtcgactct cagtacaatc tgctctgatg ccgcatagtt
     1321 aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat
     1381 ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag
     1441 gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac
     1501 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
     1561 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
     1621 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
     1681 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
     1741 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
     1801 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
     1861 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
     1921 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
     1981 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
     2041 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagagatc
     2101 cgcggccgct aatacgactc actataggga gagccgccac catggagaag aaatactcca
     2161 tcggcctgga catcggaacc aacagcgtgg gctgggccgt gatcaccgac gactataagg
     2221 tgcccagcaa gaagttcaaa gtgctgggca acacaaacag aaagagcatc aagaagaacc
     2281 tgatgggcgc cctgctgttc gactccggag agaccgccga agcaacccgg ctgaagcgca
     2341 ccgccagaag acggtacacc agaagaaaaa acaggatcag atacctgcag gagatcttcg
     2401 ccaacgagat ggccaaactg gacgatagct ttttccagcg gctggaagaa tccttcctgg
     2461 tggaggagga caagaaaaac gagagacacc ctatctttgg gaacctggcc gacgaggtgg
     2521 cctaccacag aaactacccc accatctacc acctgagaaa gaagctggcc gactcccccg
     2581 aaaaggccga cctgagactg atctacctcg ccctggccca catcatcaag tttcggggcc
     2641 acttcctgat tgaaggcaag ctgaacgccg agaactccga cgtggccaag ctgttctacc
     2701 agctgatcca gacctacaat cagctgtttg aggaaagccc tctggacgaa atcgaagtgg
     2761 acgccaaagg gatcctgtcc gcccggctga gcaagtctaa gcggctggag aagctgattg
     2821 ccgtcttccc caacgagaag aaaaacggcc tgttcggcaa cattatcgcc ctggccctgg
     2881 gcctgacccc caactttaag agcaatttcg atctgaccga ggatgccaag ctgcagctgt
     2941 ccaaggatac ctacgacgac gatctggacg agctgctggg acagatcgga gaccagtacg
     3001 ccgacctgtt cagcgccgcc aaaaacctgt ccgatgccat cctgctgagc gacatcctgc
     3061 ggagcaacag cgaagtcacc aaagcgcccc tgagcgccag catggtgaag cggtacgacg
     3121 agcaccacca ggacctggcg ctgctgaaga cactggtgag acagcagttc cccgagaagt
     3181 acgccgagat cttcaaggac gataccaaaa atggctacgc tggctacgtg ggagctgata
     3241 agaagctgcg gaagcggtcc ggcaagctgg ccaccgagga ggagttttac aagttcatca
     3301 agccgatcct ggaaaagatg gatggagccg aagagctgct ggccaagctg aaccgggacg
     3361 acctgctgag aaagcaacgg accttcgaca acggatccat ccctcaccag atccacctga
     3421 aagagctgca cgctatcctg agacggcaag aagaatttta ccccttcctg aaggagaacc
     3481 gggagaagat cgaaaagatc ctgaccttca gaatccccta ctacgtgggg ccactggccc
     3541 ggggcaatag cagattcgcc tggctgaccc ggaagtctga agaggccatc accccctgga
     3601 attttgagga ggtggtggac aaaggcgcca gcgcccagtc cttcatcgag cggatgacca
     3661 acttcgatga gcagctgcca aacaagaagg tcctgcccaa gcactctctg ctgtacgaat
     3721 attttaccgt gtacaacgaa ctgaccaaag tgaagtacgt gactgagaga atgcggaagc
     3781 ctgaattcct gtccggagag cagaagaaag ccatcgtgga cctgctgttc aagaccaaca
     3841 gaaaagtgac cgtgaaacag ctgaaggagg actacttcaa gaagatcgag tgcttcgaca
     3901 gcgtggagat tatcggagtg gaagacagat tcaacgccag cctgggcacc taccacgatc
     3961 tgctgaagat catcaaggat aaggacttcc tggacaacga ggagaacgaa gacatcctgg
     4021 aggacatcgt gctgacactg accctgttcg aggaccggga aatgatcgaa gaacggctca
     4081 aaacatacgc tcacctgttc gatgacaagg tgatgaagca gctgaagaga agacactaca
     4141 ccggctgggg ccggctgtcc cggaagatga tcaacggaat ccgggacaag cagtccggca
     4201 agacaatcct ggacttcctg aagtccgacg gcttcagcaa cagaaacttc atgcagctga
     4261 tccacgacga cagcctgacc tttaaagagg agatcgagaa agcccaggtg tccggccaag
     4321 gagactctct gcacgagcag atcgccgacc tggccggcag ccccgccatc aaaaaaggca
     4381 ttctgcagac cgtgaagatc gtggatgagc tggtgaaagt gatgggccac aagcctgaga
     4441 acatcgtgat cgagatggcc agagagaacc agaccaccac caaaggactg cagcagtcca
     4501 gggagcggaa aaagcggatc gaggaaggca tcaaggagct ggaaagccag attctgaagg
     4561 aaaaccccgt ggaaaacacc cagctgcaga acgaaaagct gtacctgtac tacctgcaga
     4621 atggcagaga tatgtacgtg gatcaggagc tggatatcaa tcggctgtcc gactacgatg
     4681 tggaccacat cgtgccccag agcttcatca aggacgactc catcgataac aaagtgctga
     4741 cccggtctgt ggagaacaga ggcaagagcg acaacgtgcc cagcgaagaa gtggtgaaga
     4801 agatgaagaa ctactggcgg cagctgctga acgccaagct gatcacccag agaaagttcg
     4861 acaacctgac caaggccgag cggggcggcc tgagcgaagc cgataaggct ggcttcatca
     4921 agcggcagct ggtggaaacc aggcagatta caaagcacgt ggcccggatc ctggacagca
     4981 gaatgaacac caagagagac aagaacgaca agcccatccg ggaggtgaag gtgatcaccc
     5041 tgaagagcaa actggtgagc gacttcagaa aagacttcca gctgtacaaa gtgcgggaca
     5101 tcaacaacta ccaccacgcc cacgacgcct acctgaacgc cgtggtgggc accgctctga
     5161 tcaagaagta ccccaagctg gagagcgagt tcgtgtacgg cgattataag gtgtacgatg
     5221 tgcggaaaat gatcgccaag agcgagcagg agatcggcaa ggccaccgcc aaacggttct
     5281 tctacagcaa catcatgaat ttcttcaaga ccgaggtgaa gctggccaac ggcgagatca
     5341 gaaagagacc tctgattgaa accaacggcg agaccggcga agtggtgtgg aacaaggaga
     5401 aggacttcgc caccgtgcgg aaggtgctgg ccatgcccca ggtgaacatc gtgaagaaga
     5461 ccgaggtgca gactggcggc ttcagcaaag agtctatcag acccaagagg aacagcgata
     5521 agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc ctgtggccca
     5581 ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc aagaaactga
     5641 agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc gagaagaatc
     5701 ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg atcatcaagc
     5761 tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg gcctctgcca
     5821 agcagctgca gaagggaaac gaactggccc tgccctccaa atatgtgaac ttcctgtacc
     5881 tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag aaacagctgt
     5941 ttgtggaaca gcacaagcac tacctggacg agatcatcga gcagatcagc gagttctcca
     6001 agagagtgat cctggccgac gctaatctgg acaaagtgct gtccgcctac aacaagcacc
     6061 gggataagcc catcagagag caggccgaga atatcatcca cctgtttacc ctgaccagac
     6121 tgggagcccc tagagccttc aagtactttg acaccaccat cgaccccaag cagtacagaa
     6181 gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc ctgtacgaga
     6241 cacggatcga cctgtctcag ctgggaggcg acggatccgg cggaggcgga agcgggaaaa
     6301 gaaccgccga cggcagcgaa ttcgagccca agaagaagag gaaagtctcg agcggaggcg
     6361 actacaaaga ccatgacggt gattataaag atcatgacat cgattacaag gatgacgatg
     6421 acaagggagg cggtggaagc ggcgcaacaa acttctctct gctgaaacaa gccggagatg
     6481 tcgaagagaa tcctggaccg atggtgagca agggcgagga gctgttcacc ggggtggtgc
     6541 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
     6601 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
     6661 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
     6721 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
     6781 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
     6841 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
     6901 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca
     6961 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
     7021 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     7081 tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg
     7141 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     7201 tggacgagct gtacaagtga ccggtcatca tcaccatcac cattgagttt aaacccgctg
     7261 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     7321 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     7381 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     7441 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
     7501 tgaggcggaa agaaccagct ggggctcgat accgtcgacc tctagctaga gcttggcgta
     7561 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     7621 acgagccgga agcataaagt gtaaagccta gggtgcctaa tgagtgagct aactcacatt
     7681 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     7741 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     7801 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     7861 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     7921 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     7981 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     8041 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     8101 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     8161 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     8221 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     8281 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     8341 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     8401 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     8461 agttggtagc tcttgatc
//
