LOCUS       sgRNA-Dual_filler(hU6_H1)-EF1a-Thy1.1-P2A-Neo        8421 bp    DNA     circular UNA 05-MAY-2025
DEFINITION  Ligation of 2 sequences.
ACCESSION   urn.local...3a-d4tn07y
VERSION     urn.local...3a-d4tn07y
KEYWORDS    .
SOURCE      
  ORGANISM  .
FEATURES             Location/Qualifiers
     primer_bind     complement(356..375)
                     /Sequence="CTTCAGCAAGCCGAGTCCTG"
                     /%GC="60.0"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="psiGIPZ_rev"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:b4-d4z3ang"">psiGIPZ_rev</a>"
                     /label="psiGIPZ_rev"
     misc_feature    454..818
                     /Color="#ffddff"
                     /modified_by="User"
                     /Transferred_From="dGag - RRL"
                     /Transferred_Similarity="100.00%"
                     /label="dGag"
     primer_bind     460..480
                     /Sequence="GCGAGAGCGTCAGTATTAAGC"
                     /%GC="52.4"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="psiGIPZ_fwd"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:be-d4z3anl"">psiGIPZ_fwd</a>"
                     /label="psiGIPZ_fwd"
     primer_bind     550..569
                     /Sequence="AACATATAGTATGGGCAAGC"
                     /%GC="40.0"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="dGAG_seq2"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:3w-d4z3aif"">dGAG_seq2</a>"
                     /label="dGAG_seq2"
     primer_bind     553..572
                     /Sequence="ATATAGTATGGGCAAGCAGG"
                     /%GC="45.0"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="dGAG_seq1"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:3r-d4z3aie"">dGAG_seq1</a>"
                     /label="dGAG_seq1"
     ORF             814..1716
                     /Genetic_code="Standard"
                     /Reading_frame=1
                     /created_by="ORF Finder"
                     /label="ORF 1 (frame 1)"
     misc_feature    963..1196
                     /label="RRE"
     primer_bind     1415..1434
                     /Sequence="GGGCAAGTTTGTGGAATTGG"
                     /%GC="50.0"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="pCas-F"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:kz-d4z3at5"">pCas-F</a>"
                     /label="pCas-F"
     primer_bind     1491..1516
                     /Sequence="CTTGGTAGGTTTAAGAATAGTTTTTG"
                     /%GC="30.8"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="Donor_sg_F"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:lo-d4z3atd"">Donor_sg_F</a>"
                     /label="Donor_sg_F"
     primer_bind     complement(1587..1599)
                     /Sequence="GGGTCCCCTCGGG"
                     /Hairpin_Tm="46.1"
                     /Tm="52.5"
                     /Self_Dimer_Tm="None"
                     /%GC="84.6"
                     /created_by="primer3"
                     /note="Derived using Geneious Prime 2020.1.1 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="pRRL-sgRNA-U6-filler-iT-EF1as-Thy1.1-P2
                     A-Neo"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; primer
                     bind (<a
                     href=""urn:local:.:d-d28sua9"">pRRL-sgRNA-U6-filler-iT-EF1
                     as-Thy1.1-P2A-Neo</a>)"
                     /label="primer bind"
     primer_bind     1600..1615
                     /Sequence="AGAGAGGGCCTATTTC"
                     /Hairpin_Tm="None"
                     /Tm="47.9"
                     /Self_Dimer_Tm="4.9"
                     /%GC="50.0"
                     /created_by="primer3"
                     /note="Derived using Geneious Prime 2020.1.1 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="pRRL-sgRNA-U6-filler-iT-EF1as-Thy1.1-P2
                     A-Neo"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; primer
                     bind (<a
                     href=""urn:local:.:d-d28sua9"">pRRL-sgRNA-U6-filler-iT-EF1
                     as-Thy1.1-P2A-Neo</a>)"
                     /label="primer bind"
     promoter        1612..1844
                     /Transferred_From="hU6, hU6 Promotor"
                     /Transferred_Similarity="100.00%"
                     /modified_by="User"
                     /label="hU6 Promoter"
     primer_bind     1732..1754
                     /Sequence="TTTCTTGGGTAGTTTGCAGTTTT"
                     /%GC="34.8"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="U6 forward"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:l4-d4z3at8"">U6 forward</a>"
                     /label="U6 forward"
     primer_bind     1774..1793
                     /Sequence="GACTATCATATGCTTACCGT"
                     /%GC="40.0"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="LKO.1 5’ (hU6 Promotor_F)"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:69-d4z3ak1"">LKO.1 5’ (hU6
                     Promotor_F)</a>"
                     /label="LKO.1 5’ (hU6 Promotor_F)"
     misc_feature    1848..2259
                     /Source="referenceToDocument: urn:local:.:2z-d4tmyne"
                     /label="Dual_filler (digested by AarI) (Insert)
                     (concatenated sequence 2)"
                     /note="Geneious type: Concatenated sequence"
     ligation        1848..1851
                     /label="Ligation"
     misc_feature    1895..1984
                     /Original_Bases="TAAAATATTATATTCTATGACCTTTATAAAATCAACCAAAA
                     TCTTCTTTTTAATAACTTTAGTATCAATAATTAGAATTTTTATGTTCCTTTTTGCAAA
                     CTTTTAATAAAAATGAGCAAAATAAAAAAACGCTAGTTTTAGTAACTCGCGTTGTTTT
                     CTTCACCTTTAATAATAGCTACTCCACCAC"
                     /label="TAAAATATTATATTCTATGACCTTTATAAAATCAACCAAAATCTTCTTTT
                     TAATAACTTTAGTATCAATAATTAGAATTTTTATGTTCCTTTTTGCAAACTTTTAATA
                     AAAATGAGCAAAATAAAAAAACGCTAGTTTTAGTAACTCGCGTTGTTTTCTTCACCTT
                     TAATAATAGCTACTCCACCAC"
                     /note="Geneious type: Editing History Replacement"
     CDS             <1895..1986
                     /created_by="julianjude"
                     /modified_by="julianjude"
                     /codon_start=3
                     /label="cr2 - Weissmann"
     promoter        1994..2208
                     /note="human H1 RNA promoter"
                     /label="H1 promoter"
     misc_difference 2256..2347
                     /label="improved tracer"
     misc_feature    2256..1851
                     /Source="referenceToDocument: urn:local:.:29-d4tmt6x"
                     /label="pRRL-sgRNA-U6-filler-improvedTracer-EF1as-Thy1.1-P
                     2A-Neo (digested by BsmBI) (Backbone) (concatenated
                     sequence 1)"
                     /note="Geneious type: Concatenated sequence"
     ligation        2256..2259
                     /label="Ligation"
     misc_feature    2390..2507
                     /label="cPPT"
     primer_bind     2451..2472
                     /Sequence="GCAACAGACATACAAACTAAAG"
                     /%GC="36.4"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="pRRL_F2"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:i7-d4z3as6"">pRRL_F2</a>"
                     /label="pRRL_F2"
     ORF             complement(2488..3054)
                     /Genetic_code="Standard"
                     /Reading_frame=1
                     /created_by="ORF Finder"
                     /label="ORF 6 (frame 1)"
     misc_feature    2551..2801
                     /ApEinfo_fwdcolor="#bc8dbf"
                     /ApEinfo_revcolor="#bc8dbf"
                     /X-Transferred_From="LentiCrisprmCherryP2Apuro-improved
                     tracer; pLentiCRISPR"
                     /X-Transferred_Similarity="100.00%"
                     /Transferred_From="pLentiCRISPR,
                     pLentisgRNA-Thy1.1-P2A-Neo, pLentisgRNA-Thy1.1-P2A-Neo
                     Copy"
                     /Transferred_Similarity="100.00%"
                     /modified_by="User"
                     /Color="#ee7700"
                     /modified_by="User"
                     /Transferred_From="EF1a short"
                     /Transferred_Similarity="100.00%"
                     /label="EF1a short variant"
                     /note="Geneious type: promoter prokaryotic"
     primer_bind     complement(2594..2613)
                     /Sequence="CAACTTCTCGGGGACTGTGG"
                     /%GC="60.0"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="EF1as_R1"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:im-d4z3asj"">EF1as_R1</a>"
                     /label="EF1as_R1"
     primer_bind     2595..2614
                     /Sequence="CACAGTCCCCGAGAAGTTGG"
                     /%GC="60.0"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="EF1as_F2"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:ih-d4z3ash"">EF1as_F2</a>"
                     /label="EF1as_F2"
     primer_bind     complement(2667..2686)
                     /Sequence="ACACGACATCACTTTCCCAG"
                     /%GC="50.0"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="EF1as_R2"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:ir-d4z3asj"">EF1as_R2</a>"
                     /label="EF1as_R2"
     primer_bind     2715..2734
                     /Sequence="GGGAGAACCGTATATAAGTG"
                     /%GC="45.0"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="EF1as_F1"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:ic-d4z3ase"">EF1as_F1</a>"
                     /label="EF1as_F1"
     ORF             2771..4153
                     /Genetic_code="Standard"
                     /Reading_frame=2
                     /created_by="ORF Finder"
                     /label="ORF 3 (frame 2)"
     CDS             2807..3292
                     /label="Thy1.1"
     primer_bind     complement(2892..2911)
                     /Sequence="GCGAAGGTTTTGGTTCACCA"
                     /%GC="50.0"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="Ty11_R2"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:hx-d4z3arx"">Ty11_R2</a>"
                     /label="Ty11_R2"
     primer_bind     complement(2893..2912)
                     /Sequence="GGCGAAGGTTTTGGTTCACC"
                     /%GC="55.0"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="Ty11_R1"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:hs-d4z3arq"">Ty11_R1</a>"
                     /label="Ty11_R1"
     primer_bind     3189..3208
                     /Sequence="TGGTCAAGTGTGGCGGCATA"
                     /%GC="55.0"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="Ty11_F1"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:hi-d4z3arc"">Ty11_F1</a>"
                     /label="Ty11_F1"
     misc            3293..3358
                     /Transferred_From="P2A"
                     /Transferred_Similarity="100.00%"
                     /annotation_group="P2A: 1 -> 66"
                     /Color="#008800"
                     /modified_by="User"
                     /label="P2A"
     primer_bind     3304..3322
                     /Sequence="CACCAACTTCAGCCTGCTG"
                     /%GC="57.9"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="M2A_F"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:pp-d4z3awb"">M2A_F</a>"
                     /label="M2A_F"
     primer_bind     3304..3322
                     /Sequence="CACCAACTTCAGCCTGCTG"
                     /%GC="57.9"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="M2A_F"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:96-d4z3am1"">M2A_F</a>"
                     /label="M2A_F"
     primer_bind     3332..3349
                     /Sequence="GGCGACGTGGAGGAGAAC"
                     /%GC="66.7"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="M2A_R"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:pu-d4z3awc"">M2A_R</a>"
                     /label="M2A_R"
     primer_bind     3332..3349
                     /Sequence="GGCGACGTGGAGGAGAAC"
                     /%GC="66.7"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="M2A_R"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:9b-d4z3am6"">M2A_R</a>"
                     /label="M2A_R"
     CDS             3359..4153
                     /Transferred_From="Neo, NeoR"
                     /Transferred_Similarity="100.00%"
                     /Color="#3333ff"
                     /modified_by="User"
                     /label="Neo"
     primer_bind     complement(3431..3449)
                     /Sequence="AGCCGATTGTCTGTTGTGC"
                     /%GC="52.6"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="NeoR"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:9l-d4z3ame"">NeoR</a>"
                     /label="NeoR"
     ORF             complement(3668..4114)
                     /Genetic_code="Standard"
                     /Reading_frame=3
                     /created_by="ORF Finder"
                     /label="ORF 7 (frame 3)"
     primer_bind     4015..4033
                     /Sequence="GGACATAGCGTTGGCTACC"
                     /%GC="57.9"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="NeoF"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:5a-d4z3aj8"">NeoF</a>"
                     /label="NeoF"
     misc_binding    4164..4752
                     /label="WPRE"
     ORF             4173..4763
                     /Genetic_code="Standard"
                     /Reading_frame=3
                     /created_by="ORF Finder"
                     /label="ORF 5 (frame 3)"
     primer_bind     4230..4249
                     /Sequence="ACGCTATGTGGATACGCTGC"
                     /%GC="55.0"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="WRE_forelement"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:bt-d4z3ao2"">WRE_forelement</a>"
                     /label="WRE_forelement"
     primer_bind     complement(4239..4258)
                     /Sequence="GGCATTAAAGCAGCGTATCC"
                     /%GC="50.0"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="WRE_reverse"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:4g-d4z3aiv"">WRE_reverse</a>"
                     /label="WRE_reverse"
     ORF             4267..4797
                     /Genetic_code="Standard"
                     /Reading_frame=1
                     /created_by="ORF Finder"
                     /label="ORF 2 (frame 1)"
     primer_bind     4525..4544
                     /Sequence="TGGGCACTGACAATTCCGTG"
                     /%GC="55.0"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="WRE_forward"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:4b-d4z3ail"">WRE_forward</a>"
                     /label="WRE_forward"
     LTR             4823..5058
                     /label="3' LTR SIN"
     primer_bind     complement(4843..4865)
                     /Sequence="AAAGCAGATCTTGTCTTCGTTGG"
                     /%GC="43.5"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="pGIPZSinR"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:b9-d4z3ank"">pGIPZSinR</a>"
                     /label="pGIPZSinR"
     polyA_signal    5135..5265
                     /label="SV40 polyadenylation signal"
     rep_origin      5296..5431
                     /created_by="User"
                     /modified_by="User"
                     /Transferred_From="SV40 ORI"
                     /Transferred_Similarity="100.00%"
                     /label="SV40 ori"
     primer_bind     complement(5450..5471)
                     /Sequence="GTAATACGACTCACTATAGGGC"
                     /%GC="45.5"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="ZUB-SEQ-T7"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:2n-d4z3ahb"">ZUB-SEQ-T7</a>"
                     /label="ZUB-SEQ-T7"
     primer_bind     complement(5500..5516)
                     /Sequence="GTTTTCCCAGTCACGAC"
                     /%GC="52.9"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="ZUB-SEQ-M13F"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:z-d4z3afy"">ZUB-SEQ-M13F</a>"
                     /label="ZUB-SEQ-M13F"
     rep_origin      5638..6093
                     /direction="RIGHT"
                     /label="F1 ori"
     primer_bind     complement(5725..5744)
                     /Sequence="AGGGAAGAAAGCGAAAGGAG"
                     /%GC="50.0"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="seqF1ori_R"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:nh-d4z3av4"">seqF1ori_R</a>"
                     /label="seqF1ori_R"
     primer_bind     complement(5775..5794)
                     /Sequence="CCCCGATTTAGAGCTTGACG"
                     /%GC="55.0"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="C3U seq primer"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:dw-d4z3apb"">C3U seq primer</a>"
                     /label="C3U seq primer"
     primer_bind     5914..5936
                     /Sequence="TGGAGTCCACGTTCTTTAATAGT"
                     /%GC="39.1"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="F1origin_forward"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:4l-d4z3aiw"">F1origin_forward</a>"
                     /label="F1origin_forward"
     promotor        6154..6182
                     /Transferred_From="Ampicillin  promoter, amp promoter"
                     /Transferred_Similarity="100.00%"
                     /annotation_group="AMP promoter: 1 -> 29"
                     /modified_by="User"
                     /label="AMP promoter"
     gene            6224..7084
                     /gene="Ampicillin"
                     /label="Ampicillin"
     ORF             6224..7084
                     /Genetic_code="Standard"
                     /Reading_frame=2
                     /created_by="ORF Finder"
                     /label="ORF 4 (frame 2)"
     primer_bind     complement(6364..6383)
                     /Sequence="TCTTACCGCTGTTGAGATCC"
                     /%GC="50.0"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="pXY_F"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:d2-d4z3aow"">pXY_F</a>"
                     /label="pXY_F"
     rep_origin      7232..7899
                     /direction="RIGHT"
                     /label="pUC ori"
     primer_bind     7713..7731
                     /Sequence="GAACAGGAGAGCGCACGAG"
                     /%GC="63.2"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="pXY_R"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:d7-d4z3aox"">pXY_R</a>"
                     /label="pXY_R"
     primer_bind     7778..7796
                     /Sequence="TTTCGCCACCTCTGACTTG"
                     /%GC="52.6"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="pUCorigin_reverse"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:4q-d4z3aj3"">pUCorigin_reverse</a>"
                     /label="pUCorigin_reverse"
     primer_bind     8231..8255
                     /Sequence="GGAAACAGCTATGACCATGATTACG"
                     /%GC="44.0"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="M13_mod_for"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:ly-d4z3atl"">M13_mod_for</a>"
                     /label="M13_mod_for"
     primer_bind     8234..8249
                     /Sequence="AACAGCTATGACCATG"
                     /%GC="43.8"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="ZUB-SEQ-M13R"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:14-d4z3ag2"">ZUB-SEQ-M13R</a>"
                     /label="ZUB-SEQ-M13R"
     promoter        8266..8285
                     /modified_by="User"
                     /X-Transferred_From="pRRL-U6-IT-Ef1as-mCherry-P2A-Puro"
                     /X-Transferred_Similarity="100.00%"
                     /Transferred_From="T3_promoter"
                     /Transferred_Similarity="100.00%"
                     /label="T3_promoter"
     primer_bind     8266..8285
                     /Sequence="AATTAACCCTCACTAAAGGG"
                     /%GC="40.0"
                     /note="Derived using Geneious Prime 2021.0.3 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="ZUB-SEQ-T3"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:2i-d4z3aha"">ZUB-SEQ-T3</a>"
                     /label="ZUB-SEQ-T3"
     LTR             8308..299
                     /label="5'LTR"
ORIGIN      
        1 tggtacgatc gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc
       61 actgaattgc cgcattgcag agatattgta tttaagtgcc tagctcgata cataaacggg
      121 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
      181 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
      241 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
      301 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga
      361 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa
      421 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc
      481 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat
      541 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg
      601 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc
      661 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc
      721 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa
      781 acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat
      841 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta
      901 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga
      961 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca
     1021 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat
     1081 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag
     1141 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg
     1201 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg
     1261 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa
     1321 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa
     1381 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac
     1441 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt
     1501 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca
     1561 ttatcgtttc agacccacct cccaaccccg aggggaccca gagagggcct atttcccatg
     1621 attccttcat atttgcatat acgatacaag gctgttagag agataattag aattaatttg
     1681 actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg
     1741 tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga
     1801 aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac cagagacggt
     1861 tgtaaatgag cacacaaaat acacatgcgt ctccgagagc taagcagaaa gctgcatagc
     1921 aagttcaaat aaggctagtc cgtacacaac ttgaaaaagt ggcagccgag tcggctgctt
     1981 ttttttcagc tccgaacgct gacgtcatca acccgctcca aggaatcgcg ggcccagtgt
     2041 cactaggcgg gaacacccag cgcgcgtgcg ccctggcagg aagatggctg tgagggacag
     2101 gggagtggcg ccctgcaata tttgcatgtc gctatgtgtt ctgggaaatc accataaacg
     2161 tgaaatgtct ttggatttgg gaatcttata agttctgtat gagaccactc tttcccagag
     2221 acgttcaatt cccggagaga cactcgaccg tctccgttta agagctatgc tggaaacagc
     2281 atagcaagtt taaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
     2341 ctttttttga attctagatc ttgagacaaa tggcagtatt catccacaat tttaaaagaa
     2401 aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca
     2461 tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca
     2521 gggacagcag agatccactt tggcgccggc tcgagtggct ccggtgcccg tcagtgggca
     2581 gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaaccggt
     2641 gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt
     2701 tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt
     2761 cgcaacgggt ttgccgccag aacacaggtg tcgtgacgcg gccaccatga acccagccat
     2821 cagcgtcgct ctcctgctct cagtcttgca ggtgtcccga gggcagaagg tgaccagcct
     2881 gacagcctgc ctggtgaacc aaaaccttcg cctggactgc cgccatgaga ataacaccaa
     2941 ggataactcc atccagcatg agttcagcct gacccgagag aagaggaagc acgtgctctc
     3001 aggcaccctc gggatacccg agcacacgta ccgctcccgc gtcaccctct ccaaccagcc
     3061 ctatatcaag gtccttaccc tagccaactt caccaccaag gatgagggcg actacttttg
     3121 tgagcttcga gtctcgggcg cgaatcccat gagctccaac aaaagtatca gtgtgtatag
     3181 agacaaactg gtcaagtgtg gcggcataag cctgctggtt cagaacacat cctggatgct
     3241 gctgctgctg ctttccctct ccctcctcca agccctggac ttcatttctc tgggcagcgg
     3301 cgccaccaac ttcagcctgc tgaagcaggc cggcgacgtg gaggagaacc ccggccccat
     3361 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     3421 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     3481 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     3541 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     3601 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     3661 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     3721 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     3781 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     3841 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg
     3901 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     3961 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     4021 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     4081 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     4141 cgagttcttc tgattaagtc gacaatcaac ctctggatta caaaatttgt gaaagattga
     4201 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
     4261 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
     4321 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
     4381 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg
     4441 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
     4501 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat
     4561 catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct
     4621 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg
     4681 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg
     4741 ccgcctcccc gcgtcgactt taagaccaat gacttacaag gcagctgtag atcttagcca
     4801 ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatct
     4861 gctttttgct tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg
     4921 ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt
     4981 gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt
     5041 gtggaaaatc tctagcagta cgtatagtag ttcatgtcat cttattattc agtatttata
     5101 acttgcaaag aaatgaatat cagagagtga gaggaacttg tttattgcag cttataatgg
     5161 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     5221 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggctct agctatcccg
     5281 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     5341 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
     5401 cagaagtagt gaggaggctt ttttggaggc ctagggacgt acccaattcg ccctatagtg
     5461 agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
     5521 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
     5581 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgggacg
     5641 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     5701 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     5761 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     5821 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     5881 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     5941 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     6001 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     6061 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
     6121 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     6181 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     6241 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     6301 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     6361 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     6421 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     6481 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     6541 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     6601 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     6661 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     6721 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     6781 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     6841 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     6901 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     6961 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     7021 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     7081 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     7141 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     7201 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     7261 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     7321 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     7381 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     7441 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     7501 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     7561 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     7621 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     7681 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     7741 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     7801 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     7861 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     7921 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     7981 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
     8041 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     8101 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
     8161 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
     8221 atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta
     8281 aagggaacaa aagctggagc tgcaagctta atgtagtctt atgcaatact cttgtagtct
     8341 tgcaacatgg taacgatgag ttagcaacat gccttacaag gagagaaaaa gcaccgtgca
     8401 tgccgattgg tggaagtaag g
//
