LOCUS       sgRNA-Dual_filler(hU6_H1/7SK_hybrid)-EF1as_Thy1.1_P2A_Neo        8298 bp    DNA     circular UNA 05-MAY-2025
DEFINITION  Gibson/Homology Cloning ligation product.
ACCESSION   urn.local...e2-em9f09f
VERSION     urn.local...e2-em9f09f
KEYWORDS    .
SOURCE      
  ORGANISM  .
FEATURES             Location/Qualifiers
     misc_feature    454..818
                     /Color="#ffddff"
                     /modified_by="User"
                     /Transferred_From="dGag - RRL"
                     /Transferred_Similarity="100.00%"
                     /label="dGag"
     misc_feature    963..1196
                     /label="RRE"
     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"
     ligation        1828..1847
                     /label="Ligation"
     misc_feature    1848..2136
                     /Source="referenceToDocument: urn:local:.:e3-em9f09f"
                     /note="Derived using Geneious Prime 2022.1.1 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="pRRL-sgRNA-U6-filler-improvedTracer-EF1
                     as-Thy1.1-P2A-Neo (digested by BsmBI) (Backbone) -
                     Dual_filler-7SK-Hybrid_pos2 (digested by AarI) Fragment 2"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0;
                     Dual_filler-7SK-Hybrid_pos2 (digested by AarI) Fragment 2
                     (concatenated sequence 2) (<a
                     href=""urn:local:.:9g-ejtckap"">pRRL-sgRNA-U6-filler-impro
                     vedTracer-EF1as-Thy1.1-P2A-Neo (digested by BsmBI)
                     (Backbone) - Dual_filler-7SK-Hybrid_pos2 (digested by
                     AarI) Fragment 2</a>)"
                     /label="Dual_filler-7SK-Hybrid_pos2 (digested by AarI)
                     Fragment 2 (concatenated sequence 2)"
                     /note="Geneious type: Concatenated sequence"
     primer_bind     1848..1868
                     /created_by="Biochem_Jannis"
                     /Sequence="CACCAGAGACGGTTGTAAATG"
                     /Tm_(binding_region)="56.9"
                     /Tm_(with_extension)="65.2"
                     /%GC_(binding_region)="47.6"
                     /%GC_(with_extension)="43.9"
                     /Hairpin_Tm_(with_extension)="32.0"
                     /Self_Dimer_Tm_(with_extension)="None"
                     /Extension="ATCTTGTGGAAAGGACGAAA"
                     /modified_by="Biochem_Jannis"
                     /label="Gibson_for"
     CDS             <1895..1986
                     /created_by="julianjude"
                     /modified_by="julianjude"
                     /codon_start=3
                     /label="cr2 - Weissmann"
     promoter        1994..>2093
                     /created_by="Philipp"
                     /modified_by="Philipp"
                     /Color="#baff00"
                     /modified_by="Biochem_Jannis"
                     /label="H1/7SK hybrid (H1 M11)"
     primer_bind     complement(2118..2136)
                     /created_by="Biochem_Jannis"
                     /Sequence="AAACGGAGACGGTCGAGTG"
                     /Tm_(binding_region)="59.7"
                     /Tm_(with_extension)="66.4"
                     /%GC_(binding_region)="57.9"
                     /%GC_(with_extension)="51.3"
                     /Hairpin_Tm_(with_extension)="37.6"
                     /Self_Dimer_Tm_(with_extension)="11.8"
                     /Extension="CTGTTTCCAGCATAGCTCTT"
                     /modified_by="Biochem_Jannis"
                     /label="Gibson_rev"
     misc_feature    2136^2137
                     /Original_Bases="CGG"
                     /label="CGG"
                     /note="Geneious type: Editing History Deletion"
     misc_difference <2137..2224
                     /label="improved tracer"
     ligation        2137..2156
                     /label="Ligation"
     misc_feature    2137..1847
                     /Source="referenceToDocument: urn:local:.:jg-ej3u46r"
                     /label="pRRL-sgRNA-U6-filler-improvedTracer-EF1as-Thy1.1-P
                     2A-Neo (digested by BsmBI) (Backbone) (concatenated
                     sequence 1)"
                     /note="Geneious type: Concatenated sequence"
     misc_feature    2267..2384
                     /label="cPPT"
     misc_feature    2428..2678
                     /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"
     CDS             2684..3169
                     /label="Thy1.1"
     misc            3170..3235
                     /Transferred_From="P2A"
                     /Transferred_Similarity="100.00%"
                     /annotation_group="P2A: 1 -> 66"
                     /Color="#008800"
                     /modified_by="User"
                     /label="P2A"
     CDS             3236..4030
                     /Transferred_From="Neo, NeoR"
                     /Transferred_Similarity="100.00%"
                     /Color="#3333ff"
                     /modified_by="User"
                     /label="Neo"
     misc_binding    4041..4629
                     /label="WPRE"
     LTR             4700..4935
                     /label="3' LTR SIN"
     polyA_signal    5012..5142
                     /label="SV40 polyadenylation signal"
     rep_origin      5173..5308
                     /created_by="User"
                     /modified_by="User"
                     /Transferred_From="SV40 ORI"
                     /Transferred_Similarity="100.00%"
                     /label="SV40 ori"
     rep_origin      5515..5970
                     /direction="RIGHT"
                     /label="F1 ori"
     promotor        6031..6059
                     /Transferred_From="Ampicillin  promoter, amp promoter"
                     /Transferred_Similarity="100.00%"
                     /annotation_group="AMP promoter: 1 -> 29"
                     /modified_by="User"
                     /label="AMP promoter"
     gene            6101..6961
                     /gene="Ampicillin"
                     /label="Ampicillin"
     rep_origin      7109..7776
                     /direction="RIGHT"
                     /label="pUC ori"
     promoter        8143..8162
                     /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"
     LTR             8185..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 tccatattta gcatgtcgct atgtgttctg ggaaacttga cctaagtgta
     2041 aagttgagat ttccttcagg tttatatagt tctgtatgag accactcttt cccagagacg
     2101 ttcaattccc ggagagacac tcgaccgtct ccgtttaaga gctatgctgg aaacagcata
     2161 gcaagtttaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
     2221 tttttgaatt ctagatcttg agacaaatgg cagtattcat ccacaatttt aaaagaaaag
     2281 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
     2341 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
     2401 acagcagaga tccactttgg cgccggctcg agtggctccg gtgcccgtca gtgggcagag
     2461 cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg aaccggtgcc
     2521 tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt
     2581 cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc
     2641 aacgggtttg ccgccagaac acaggtgtcg tgacgcggcc accatgaacc cagccatcag
     2701 cgtcgctctc ctgctctcag tcttgcaggt gtcccgaggg cagaaggtga ccagcctgac
     2761 agcctgcctg gtgaaccaaa accttcgcct ggactgccgc catgagaata acaccaagga
     2821 taactccatc cagcatgagt tcagcctgac ccgagagaag aggaagcacg tgctctcagg
     2881 caccctcggg atacccgagc acacgtaccg ctcccgcgtc accctctcca accagcccta
     2941 tatcaaggtc cttaccctag ccaacttcac caccaaggat gagggcgact acttttgtga
     3001 gcttcgagtc tcgggcgcga atcccatgag ctccaacaaa agtatcagtg tgtatagaga
     3061 caaactggtc aagtgtggcg gcataagcct gctggttcag aacacatcct ggatgctgct
     3121 gctgctgctt tccctctccc tcctccaagc cctggacttc atttctctgg gcagcggcgc
     3181 caccaacttc agcctgctga agcaggccgg cgacgtggag gagaaccccg gccccatgat
     3241 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     3301 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     3361 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga
     3421 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     3481 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     3541 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     3601 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     3661 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     3721 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga
     3781 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     3841 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     3901 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     3961 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     4021 gttcttctga ttaagtcgac aatcaacctc tggattacaa aatttgtgaa agattgactg
     4081 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
     4141 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc
     4201 tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
     4261 ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga
     4321 ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct
     4381 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat
     4441 cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct
     4501 gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc
     4561 tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg
     4621 cctccccgcg tcgactttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt
     4681 tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac aagatctgct
     4741 ttttgcttgt actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
     4801 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
     4861 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
     4921 gaaaatctct agcagtacgt atagtagttc atgtcatctt attattcagt atttataact
     4981 tgcaaagaaa tgaatatcag agagtgagag gaacttgttt attgcagctt ataatggtta
     5041 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     5101 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggctctagc tatcccgccc
     5161 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     5221 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag
     5281 aagtagtgag gaggcttttt tggaggccta gggacgtacc caattcgccc tatagtgagt
     5341 cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
     5401 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag
     5461 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc
     5521 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     5581 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     5641 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     5701 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     5761 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     5821 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     5881 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     5941 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
     6001 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     6061 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     6121 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     6181 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     6241 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     6301 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     6361 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     6421 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     6481 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     6541 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     6601 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     6661 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     6721 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     6781 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     6841 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     6901 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     6961 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     7021 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     7081 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     7141 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     7201 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     7261 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     7321 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     7381 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     7441 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     7501 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     7561 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     7621 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     7681 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     7741 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     7801 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     7861 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
     7921 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     7981 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
     8041 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
     8101 tcacacagga aacagctatg accatgatta cgccaagcgc gcaattaacc ctcactaaag
     8161 ggaacaaaag ctggagctgc aagcttaatg tagtcttatg caatactctt gtagtcttgc
     8221 aacatggtaa cgatgagtta gcaacatgcc ttacaaggag agaaaaagca ccgtgcatgc
     8281 cgattggtgg aagtaagg
//
