LOCUS       pXJ197_AAV_SRT_gRNA_dir 6891 bp ds-DNA     circular     01-DEC-2023
DEFINITION  .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
COMMENT     9Backbone without insert 4.4kb.  Imported using the Genbank
            importer. File name: addgene-plasmid-154889-sequence-306065.gbk
            piggyBac self-reporting transposon.
FEATURES             Location/Qualifiers
     CDS             complement(171..1031)
                     /label="AmpR"
     primer_bind     794..813
                     /label="Amp-R"
     promoter        complement(1032..1095)
                     /label="AmpR promoter"
     misc_feature    1475..1491
                     /label="M13f40-genewiz"
     primer_bind     1480..1502
                     /label="M13/pUC Forward"
     primer_bind     1494..1511
                     /label="M13 Forward"
     primer_bind     1495..1511
                     /label="M13 fwd"
     promoter        1522..1540
                     /label="T7 promoter"
     primer_bind     1522..1541
                     /label="T7"
     repeat_region   1577..1706
                     /label="AAV2 ITR"
     repeat_region   1853..1915
                     /label="piggyBac right (3') inverted repeat"
     misc_feature    2150..2390
                     /label="U6 prom"
     misc_feature    2399..2416
                     /label="empty guide"
     misc_feature    2417..2492
                     /label="gRNA scaffold"
     misc_feature    2449..2469
                     /label="5' kit"
     misc_feature    2493..2514
                     /label="direct capture 1 (10x genomics)"
     promoter        2539..3710
                     /label="EF1Alpha"
     misc_feature    3675..3719
                     /label="fwd overhang"
     misc_feature    3720..3722
                     /label="Kozak Sequence"
     primer          3720..3739
                     /label="fwd overhang"
                     /note="sequence: atggtgagcaagggcgagga"
     exon            3720..4436
                     /label="GFP"
     CDS             3720..4436
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK"
     primer          4012..4030
                     /label="GFP-qPCR-1 F"
                     /note="sequence: ccatcttcttcaaggacga"
     primer          complement(4142..4161)
                     /label="GFP-qPCR-1 R"
                     /note="sequence: tgttgtagttgtactccagc"
     primer          4160..4179
                     /label="GFP-qPCR-2 F"
                     /note="sequence: cagccacaacgtctatatca"
     primer          complement(4262..4279)
                     /label="GFP-qPCR-2 R"
                     /note="sequence: gtgttctgctggtagtgg"
     primer          complement(4414..4436)
                     /label="rev overhang"
                     /note="sequence: cttgtacagctcgtccatgccga"
     misc_feature    4437..4703
                     /label="KASH-tn5confimred"
     CDS             4437..4703
                     /label="Translation 4437-4703"
                     /translation="SGLRSREEEEETDSRMPHLDSPGSSQPRRSFLSRVIRAALPLQLLLLLLLLLACLLPASEDDYSCTQANNFARSFYPMLRYTNGPPPT*"
     primer          4524..4543
                     /label="qPCR-3-F"
                     /note="sequence: TCCTTCCTCTCAAGGGTGAT"
     primer          complement(4641..4659)
                     /label="qPCR-3-R"
                     /note="sequence: AGGATCGGGCAAAGTTGTT"
     misc_feature    4710..4738
                     /label="rev overhang"
     misc_feature    4753..5229
                     /label="hGH polyA signal"
     misc_feature    complement(4795..4814)
                     /label="hGH polyA rev for Sanger Genewiz"
     misc_feature    5242..5261
                     /label="salazar rev1"
     misc_feature    5262..5290
                     /label="AS2_gibson_flanking"
     repeat_region   5550..5584
                     /label="piggyBac left (5') inverted repeat"
     repeat_region   5892..6021
                     /label="AAV2 ITR"
     primer_bind     complement(6149..6166)
                     /label="L4440"
     terminator      6203..6246
                     /label="rrnB T1 terminator"
     rep_origin      complement(6303..6891)
                     /label="ori"
     primer_bind     complement(6383..6402)
                     /label="pBR322ori-F"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      301 atgataccgc gagaaccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
      721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1081 atgagcggat acatatttga agctaataac aggcctgctg gtaatcgcag gcctttttat
     1141 ttggtgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     1201 acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     1261 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     1321 gagatagggt tgagtggccg ctacagggcg ctcccattcg ccattcaggc tgcgcaactg
     1381 ttgggaaggg cgtttcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
     1441 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa
     1501 cgacggccag tgagcgcgac gtaatacgac tcactatagg gcgaattgta ggcggaaggc
     1561 cgtcaaggcc gcatggctgc gcgctcgctc gctcactgag gccgcccggg caaagcccgg
     1621 gcgtcgggcg acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt
     1681 ggccaactcc atcactaggg gttcctattg acgtcagatc gctaaataat gacgtatgtt
     1741 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
     1801 actgcccact tggcagtaca tcaagtgtat catacgtcaa ggccgcattt aaccctagaa
     1861 agataatcat attgtgacgt acgttaaaga taatcatgtg taaaattgac gcatgtgttt
     1921 tatcggtctg tatatcgagg tttatttatt aatttgaata gatattaagt tttattatat
     1981 ttacacttac atactaataa taaattcaac aaacaattta tttatgttta tttatttatt
     2041 aaaaaaaaca aaaactcaaa atttcttcta taaagtaaca aaacttttat cgaattcctg
     2101 cagcccagct tgatcttagg tccctcgacg aattctctag agcacgcgtg agggcctatt
     2161 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat
     2221 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt
     2281 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt
     2341 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccGG
     2401 AAGAGCGAGC TCTTCTGTTT TAGAGCTAGA AATAGCAAGT TAAAATAAGG CTAGTCCGTT
     2461 ATCAACTTGA AAAAGTGGCA CCGAGTCGGT GCGCTTTAAG GCCGGTCCTA GCAAtttttt
     2521 tctagactgc agagctgcGT GCCCGTCAGT GGGCAGAGCG CACATCGCCC ACAGTCCCCG
     2581 AGAAGTTGGG GGGAGGGGTC GGCAATTGAA CCGGTGCCTA GAGAAGGTGG CGCGGGGTAA
     2641 ACTGGGAAAG TGATGTCGTG TACTGGCTCC GCCTTTTTCC CGAGGGTGGG GGAGAACCGT
     2701 ATATAAGTGC AGTAGTCGCC GTGAACGTTC TTTTTCGCAA CGGGTTTGCC GCCAGAACAC
     2761 AGGTAAGTGC CGTGTGTGGT TCCCGCGGGC CTGGCCTCTT TACGGGTTAT GGCCCTTGCG
     2821 TGCCTTGAAT TACTTCCACC TGGCTGCAGT ACGTGATTCT TGATCCCGAG CTTCGGGTTG
     2881 GAAGTGGGTG GGAGAGTTCG AGGCCTTGCG CTTAAGGAGC CCCTTCGCCT CGTGCTTGAG
     2941 TTGAGGCCTG GCCTGGGCGC TGGGGCCGCC GCGTGCGAAT CTGGTGGCAC CTTCGCGCCT
     3001 GTCTCGCTGC TTTCGATAAG TCTCTAGCCA TTTAAAATTT TTGATGACCT GCTGCGACGC
     3061 TTTTTTTCTG GCAAGATAGT CTTGTAAATG CGGGCCAAGA TCTGCACACT GGTATTTCGG
     3121 TTTTTGGGGC CGCGGGCGGC GACGGGGCCC GTGCGTCCCA GCGCACATGT TCGGCGAGGC
     3181 GGGGCCTGCG AGCGCGGCCA CCGAGAATCG GACGGGGGTA GTCTCAAGCT GGCCGGCCTG
     3241 CTCTGGTGCC TGGCCTCGCG CCGCCGTGTA TCGCCCCGCC CTGGGCGGCA AGGCTGGCCC
     3301 GGTCGGCACC AGTTGCGTGA GCGGAAAGAT GGCCGCTTCC CGGCCCTGCT GCAGGGAGCT
     3361 CAAAATGGAG GACGCGGCGC TCGGGAGAGC GGGCGGGTGA GTCACCCACA CAAAGGAAAA
     3421 GGGCCTTTCC GTCCTCAGCC GTCGCTTCAT GTGACTCCAC GGAGTACCGG GCGCCGTCCA
     3481 GGCACCTCGA TTAGTTCTCG cGCTTTTGGA GTACGTCGTC TTTAGGTTGG GGGGAGGGGT
     3541 TTTATGCGAT GGAGTTTCCC CACACTGAGT GGGTGGAGAC TGAAGTTAGG CCAGCTTGGC
     3601 ACTTGATGTA ATTCTCCTTG GAATTTGCCC TTTTTGAGTT TGGATCTTGG TTCATTCTCA
     3661 AGCCTCAGAC AGTGGTTCAA AGTTTTTTTC TTCCATTTCA GGTGTCGTGA gCTAGCACCa
     3721 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     3781 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     3841 gcaagctgac cctgaagttc atttgcacca ccggcaagct gcccgtgccc tggcccaccc
     3901 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
     3961 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     4021 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     4081 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     4141 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     4201 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     4261 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     4321 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     4381 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagTCCG
     4441 GACTCAGATC TCGAGAGGAG GAGGAGGAGA CAGACAGCAG GATGCCCCAC CTCGACAGCC
     4501 CCGGCAGCTC CCAGCCGAGA CGCTCCTTCC TCTCAAGGGT GATCAGGGCA GCGCTACCGT
     4561 TGCAGCTGCT TCTGCTGCTG CTGCTGCTCC TGGCCTGCCT GCTACCTGCC TCTGAAGATG
     4621 ACTACAGCTG CACCCAGGCC AACAACTTTG CCCGATCCTT CTACCCCATG CTGCGGTACA
     4681 CCAACGGGCC ACCTCCCACC TAGgaattcg atatcaagct tatcgatacc gagcgctgct
     4741 cgagagatct acgggtggca tccctgtgac ccctccccag tgcctctcct ggccctggaa
     4801 gttgccactc cagtgcccac cagccttgtc ctaataaaat taagttgcat cattttgtct
     4861 gactaggtgt ccttctataa tattatgggg tggagggggg tggtatggag caaggggcaa
     4921 gttgggaaga caacctgtag ggcctgcggg gtctattggg aaccaagctg gagtgcagtg
     4981 gcacaatctt ggctcactgc aatctccgcc tcctgggttc aagcgattct cctgcctcag
     5041 cctcccgagt tgttgggatt ccaggcatgc atgaccaggc tcagctaatt tttgtttttt
     5101 tggtagagac ggggtttcac catattggcc aggctggtct ccaactccta atctcaggtg
     5161 atctacccac cttggcctcc caaattgctg ggattacagg cgtgaaccac tgctcccttc
     5221 cctgtccttc tgattttgta ggtaaccacg tgcggaccga gcgccggcgc gataagctat
     5281 ctataacaag aaaatatata tataataagt tatcacgtaa gtagaacatg aaataacaat
     5341 ataattatcg tatgagttaa atcttaaaag tcacgtaaaa gataatcatg cgtcattttg
     5401 actcacgcgg tcgttatagt tcaaaatcag tgacacttac cgcattgaca agcacgcctc
     5461 acgggagctc caagcggcga ctgagatgtc ctaaatgcac agcgacggat tcgcgctatt
     5521 tagaaagaga gagcaatatt tcaagaatgc atgcgtcaat tttacgcaga ctatctttct
     5581 agggttaaga gctcctgaga tgcaggtaca tcccactgat gagtcccaaa taggacgaaa
     5641 cgcgcttcgg tgcgtctggg attccactgc tatccacgcg gccgcacggc attacgttag
     5701 acgtctaggc cacaccaccc tgaaggcgcc tgctcgcctc tgatctgttg aagctaagca
     5761 gggtcggtcc tggttagtac ttggatggga ctccgcctgg taatagccgg taccgtaggc
     5821 ttgcatggtc atagctgttt ccttgcgctg gggatgcggt gggctctata gagagtcgat
     5881 cgtgtgatcg aaggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc
     5941 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc
     6001 agtgagcgag cgagcgcgca gcatgtagag actgggcctc atgggccttc cgcctatcac
     6061 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaacatggt catagctgtt
     6121 tccttgcgta ttgggcgctc tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     6181 gggtaaagcc tggggtgcct aaataaaacg aaaggcccag tcttccgact gagcctttcg
     6241 ttttattgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     6301 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     6361 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     6421 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     6481 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     6541 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     6601 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     6661 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     6721 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     6781 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     6841 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca a
//