LOCUS       pXJ220_AAV_SRT_gRNA_EF1 6869 bp ds-DNA     circular     13-MAY-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"
     promoter        2517..3688
                     /label="EF1Alpha"
     misc_feature    3653..3697
                     /label="fwd overhang"
     misc_feature    3698..3700
                     /label="Kozak Sequence"
     primer          3698..3717
                     /label="fwd overhang"
                     /note="sequence: atggtgagcaagggcgagga"
     exon            3698..4414
                     /label="GFP"
     CDS             3698..4414
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK"
     primer          3990..4008
                     /label="GFP-qPCR-1 F"
                     /note="sequence: ccatcttcttcaaggacga"
     primer          complement(4120..4139)
                     /label="GFP-qPCR-1 R"
                     /note="sequence: tgttgtagttgtactccagc"
     primer          4138..4157
                     /label="GFP-qPCR-2 F"
                     /note="sequence: cagccacaacgtctatatca"
     primer          complement(4240..4257)
                     /label="GFP-qPCR-2 R"
                     /note="sequence: gtgttctgctggtagtgg"
     primer          complement(4392..4414)
                     /label="rev overhang"
                     /note="sequence: cttgtacagctcgtccatgccga"
     misc_feature    4415..4681
                     /label="KASH-tn5confimred"
     CDS             4415..4681
                     /label="Translation 4415-4681"
                     /translation="SGLRSREEEEETDSRMPHLDSPGSSQPRRSFLSRVIRAALPLQLLLLLLLLLACLLPASEDDYSCTQANNFARSFYPMLRYTNGPPPT*"
     primer          4502..4521
                     /label="qPCR-3-F"
                     /note="sequence: TCCTTCCTCTCAAGGGTGAT"
     primer          complement(4619..4637)
                     /label="qPCR-3-R"
                     /note="sequence: AGGATCGGGCAAAGTTGTT"
     misc_feature    4688..4716
                     /label="rev overhang"
     misc_feature    4731..5207
                     /label="hGH polyA signal"
     misc_feature    complement(4773..4792)
                     /label="hGH polyA rev for Sanger Genewiz"
     misc_feature    5220..5239
                     /label="salazar rev1"
     misc_feature    5240..5268
                     /label="AS2_gibson_flanking"
     repeat_region   5528..5562
                     /label="piggyBac left (5') inverted repeat"
     repeat_region   5870..5999
                     /label="AAV2 ITR"
     primer_bind     complement(6127..6144)
                     /label="L4440"
     terminator      6181..6224
                     /label="rrnB T1 terminator"
     rep_origin      complement(6281..6869)
                     /label="ori"
     primer_bind     complement(6361..6380)
                     /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 GCtttttttc tagactgcag agctgcGTGC
     2521 CCGTCAGTGG GCAGAGCGCA CATCGCCCAC AGTCCCCGAG AAGTTGGGGG GAGGGGTCGG
     2581 CAATTGAACC GGTGCCTAGA GAAGGTGGCG CGGGGTAAAC TGGGAAAGTG ATGTCGTGTA
     2641 CTGGCTCCGC CTTTTTCCCG AGGGTGGGGG AGAACCGTAT ATAAGTGCAG TAGTCGCCGT
     2701 GAACGTTCTT TTTCGCAACG GGTTTGCCGC CAGAACACAG GTAAGTGCCG TGTGTGGTTC
     2761 CCGCGGGCCT GGCCTCTTTA CGGGTTATGG CCCTTGCGTG CCTTGAATTA CTTCCACCTG
     2821 GCTGCAGTAC GTGATTCTTG ATCCCGAGCT TCGGGTTGGA AGTGGGTGGG AGAGTTCGAG
     2881 GCCTTGCGCT TAAGGAGCCC CTTCGCCTCG TGCTTGAGTT GAGGCCTGGC CTGGGCGCTG
     2941 GGGCCGCCGC GTGCGAATCT GGTGGCACCT TCGCGCCTGT CTCGCTGCTT TCGATAAGTC
     3001 TCTAGCCATT TAAAATTTTT GATGACCTGC TGCGACGCTT TTTTTCTGGC AAGATAGTCT
     3061 TGTAAATGCG GGCCAAGATC TGCACACTGG TATTTCGGTT TTTGGGGCCG CGGGCGGCGA
     3121 CGGGGCCCGT GCGTCCCAGC GCACATGTTC GGCGAGGCGG GGCCTGCGAG CGCGGCCACC
     3181 GAGAATCGGA CGGGGGTAGT CTCAAGCTGG CCGGCCTGCT CTGGTGCCTG GCCTCGCGCC
     3241 GCCGTGTATC GCCCCGCCCT GGGCGGCAAG GCTGGCCCGG TCGGCACCAG TTGCGTGAGC
     3301 GGAAAGATGG CCGCTTCCCG GCCCTGCTGC AGGGAGCTCA AAATGGAGGA CGCGGCGCTC
     3361 GGGAGAGCGG GCGGGTGAGT CACCCACACA AAGGAAAAGG GCCTTTCCGT CCTCAGCCGT
     3421 CGCTTCATGT GACTCCACGG AGTACCGGGC GCCGTCCAGG CACCTCGATT AGTTCTCGcG
     3481 CTTTTGGAGT ACGTCGTCTT TAGGTTGGGG GGAGGGGTTT TATGCGATGG AGTTTCCCCA
     3541 CACTGAGTGG GTGGAGACTG AAGTTAGGCC AGCTTGGCAC TTGATGTAAT TCTCCTTGGA
     3601 ATTTGCCCTT TTTGAGTTTG GATCTTGGTT CATTCTCAAG CCTCAGACAG TGGTTCAAAG
     3661 TTTTTTTCTT CCATTTCAGG TGTCGTGAgC TAGCACCatg gtgagcaagg gcgaggagct
     3721 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     3781 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
     3841 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg
     3901 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     3961 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     4021 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     4081 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     4141 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
     4201 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     4261 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct
     4321 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     4381 cgggatcact ctcggcatgg acgagctgta caagTCCGGA CTCAGATCTC GAGAGGAGGA
     4441 GGAGGAGACA GACAGCAGGA TGCCCCACCT CGACAGCCCC GGCAGCTCCC AGCCGAGACG
     4501 CTCCTTCCTC TCAAGGGTGA TCAGGGCAGC GCTACCGTTG CAGCTGCTTC TGCTGCTGCT
     4561 GCTGCTCCTG GCCTGCCTGC TACCTGCCTC TGAAGATGAC TACAGCTGCA CCCAGGCCAA
     4621 CAACTTTGCC CGATCCTTCT ACCCCATGCT GCGGTACACC AACGGGCCAC CTCCCACCTA
     4681 Ggaattcgat atcaagctta tcgataccga gcgctgctcg agagatctac gggtggcatc
     4741 cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca gtgcccacca
     4801 gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc ttctataata
     4861 ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca acctgtaggg
     4921 cctgcggggt ctattgggaa ccaagctgga gtgcagtggc acaatcttgg ctcactgcaa
     4981 tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg ttgggattcc
     5041 aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagagacgg ggtttcacca
     5101 tattggccag gctggtctcc aactcctaat ctcaggtgat ctacccacct tggcctccca
     5161 aattgctggg attacaggcg tgaaccactg ctcccttccc tgtccttctg attttgtagg
     5221 taaccacgtg cggaccgagc gccggcgcga taagctatct ataacaagaa aatatatata
     5281 taataagtta tcacgtaagt agaacatgaa ataacaatat aattatcgta tgagttaaat
     5341 cttaaaagtc acgtaaaaga taatcatgcg tcattttgac tcacgcggtc gttatagttc
     5401 aaaatcagtg acacttaccg cattgacaag cacgcctcac gggagctcca agcggcgact
     5461 gagatgtcct aaatgcacag cgacggattc gcgctattta gaaagagaga gcaatatttc
     5521 aagaatgcat gcgtcaattt tacgcagact atctttctag ggttaagagc tcctgagatg
     5581 caggtacatc ccactgatga gtcccaaata ggacgaaacg cgcttcggtg cgtctgggat
     5641 tccactgcta tccacgcggc cgcacggcat tacgttagac gtctaggcca caccaccctg
     5701 aaggcgcctg ctcgcctctg atctgttgaa gctaagcagg gtcggtcctg gttagtactt
     5761 ggatgggact ccgcctggta atagccggta ccgtaggctt gcatggtcat agctgtttcc
     5821 ttgcgctggg gatgcggtgg gctctataga gagtcgatcg tgtgatcgaa ggaaccccta
     5881 gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca
     5941 aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc
     6001 atgtagagac tgggcctcat gggccttccg cctatcactg cccgctttcc agtcgggaaa
     6061 cctgtcgtgc cagctgcatt aacatggtca tagctgtttc cttgcgtatt gggcgctctc
     6121 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg gtaaagcctg gggtgcctaa
     6181 ataaaacgaa aggcccagtc ttccgactga gcctttcgtt ttattgagca aaaggccagc
     6241 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     6301 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     6361 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     6421 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     6481 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     6541 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     6601 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     6661 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     6721 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     6781 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     6841 agattacgcg cagaaaaaaa ggatctcaa
//