LOCUS       XJ385_pAAV-PBIR-hU6-gRN 6851 bp ds-DNA     circular     08-JUN-2024
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"
     primer          2111..2169
                     /label="F GG Gib primer"
                     /note="sequence: tgatcttaggtccctcgacgaattctctagagcacgcgtgagggcctatttcccatgat"
     misc_feature    2150..2390
                     /label="U6 prom"
     primer          2152..2173
                     /label="gRNA distribution F"
                     /note="sequence: gggcctatttcccatgattcct"
     misc_feature    2399..2416
                     /label="empty guide"
     misc_feature    2417..2492
                     /label="gRNA scaffold"
     misc_feature    2449..2469
                     /label="5' kit"
     primer          complement(2467..2488)
                     /label="gRNA distribution R"
                     /note="sequence: cgactcggtgccactttttcaa"
     promoter        2517..3688
                     /label="EF1Alpha"
     misc_feature    3653..3694
                     /label="fwd overhang"
     primer          3664..3714
                     /label="F primer XJ385 mScarlet"
                     /note="sequence: TTTTCTTCCATTTCAGGTGTCGTGAgCTAGCgccaccatggtgagcaaggg"
     primer          3666..3693
                     /label="Nhe_F1"
                     /note="sequence: TTCTTCCATTTCAGGTGTCGTGAgCTAG"
     regulatory      3695..3700
                     /label="Kozak sequence"
                     /note="note: vertebrate consensus sequence for strong initiation of translation (Kozak, 1987) regulatory_class: other: "
     CDS             3701..4396
                     /label="Translation 3701-4396"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTGGMDELYK"
     CDS             3701..4396
                     /label="Translation 3701-4396"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTGGMDELYK"
     misc_feature    3701..4396
                     /label="mScarlet"
     CAAT_signal     3702..3703
                     /label="TS"
     CDS             3704..4396
                     /label="mScarlet"
                     /note="codon_start: 1 product: bright monomeric red fluorescent protein evolved from a synthetic template (Bindels et al., 2016) transl_table: 1 translation: VSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTGGMDELYK"
     misc_feature    complement(4374..4393)
                     /label="ggready-R"
     primer          complement(4377..4427)
                     /label="XJ857-GFP-KASH-R"
                     /note="sequence: CCTCCTCCTCCTCTCGAGATCTGAGTCCGGActtgtacagctcgtccatgc"
     misc_feature    4397..4663
                     /label="KASH-tn5confimred"
     CDS             4397..4663
                     /label="Translation 4397-4663"
                     /translation="SGLRSREEEEETDSRMPHLDSPGSSQPRRSFLSRVIRAALPLQLLLLLLLLLACLLPASEDDYSCTQANNFARSFYPMLRYTNGPPPT*"
     primer          4484..4503
                     /label="qPCR-3-F"
                     /note="sequence: TCCTTCCTCTCAAGGGTGAT"
     primer          complement(4601..4619)
                     /label="qPCR-3-R"
                     /note="sequence: AGGATCGGGCAAAGTTGTT"
     primer          complement(4645..4661)
                     /label="KASH_R1"
                     /note="sequence: taatccagaggttgattgtcgacttaacgcgttcaGGTGGGAGGTGGCCCG"
     misc_feature    4648..4672
                     /label="fwd overhang BC"
     misc_feature    4670..4675
                     /label="EcoRV cut"
     misc_feature    4670..4698
                     /label="rev overhang"
     misc_feature    4673..4696
                     /label="rev overhang BC"
     misc_feature    4682..4687
                     /label="Bsu15I cut"
     misc_feature    4713..5189
                     /label="hGH polyA signal"
     misc_feature    complement(4755..4774)
                     /label="hGH polyA rev for Sanger Genewiz"
     misc_feature    5202..5221
                     /label="salazar rev1"
     misc_feature    5222..5250
                     /label="AS2_gibson_flanking"
     repeat_region   5510..5544
                     /label="piggyBac left (5') inverted repeat"
     repeat_region   5852..5981
                     /label="AAV2 ITR"
     primer_bind     complement(6109..6126)
                     /label="L4440"
     terminator      6163..6206
                     /label="rrnB T1 terminator"
     rep_origin      complement(6263..6851)
                     /label="ori"
     primer_bind     complement(6343..6362)
                     /label="pBR322ori-F"
     primer          6804..1
                     /label="EP04"
                     /note="sequence: CTTCCATTTCAGGTGTCGTGAggcaagtttgtacaaaaaagcaggctcg"
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 TAGCgccacc atggtgagca agggcgaggc
     3721 agtgatcaag gagttcatgc ggttcaaggt gcacatggag ggctccatga acggccacga
     3781 gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct
     3841 gaaggtgacc aagggtggcc ccctgccctt ctcctgggac atcctgtccc ctcagttcat
     3901 gtacggctcc agggccttca ccaagcaccc cgccgacatc cccgactact ataagcagtc
     3961 cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgccgtgac
     4021 cgtgacccag gacacctccc tggaggacgg caccctgatc tacaaggtga agctccgcgg
     4081 caccaacttc cctcctgacg gccccgtaat gcagaagaag acaatgggct gggaagcgtc
     4141 caccgagcgg ttgtaccccg aggacggcgt gctgaagggc gacattaaga tggccctgcg
     4201 cctgaaggac ggcgggcgct acctggcgga cttcaagacc acctacaagg ccaagaagcc
     4261 cgtgcagatg cccggcgcct acaacgtgga ccgcaagttg gacatcacct cccacaacga
     4321 ggactacacc gtggtggaac agtacgaacg ctccgagggc cgccactcca ccggcggcat
     4381 ggacgagctg tacaagtccg gactcagatc tcgagaggag gaggaggaga cagacagcag
     4441 gatgccccac ctcgacagcc ccggcagctc ccagccgaga cgctccttcc tctcaagggt
     4501 gatcagggca gcgctaccgt tgcagctgct tctgctgctg ctgctgctcc tggcctgcct
     4561 gctacctgcc tctgaagatg actacagctg cacccaggcc aacaactttg cccgatcctt
     4621 ctaccccatg ctgcggtaca ccaacgggcc acctcccacc taggaattcg atatcaagct
     4681 tatcgatacc gagcgctgct cgagagatct acgggtggca tccctgtgac ccctccccag
     4741 tgcctctcct ggccctggaa gttgccactc cagtgcccac cagccttgtc ctaataaaat
     4801 taagttgcat cattttgtct gactaggtgt ccttctataa tattatgggg tggagggggg
     4861 tggtatggag caaggggcaa gttgggaaga caacctgtag ggcctgcggg gtctattggg
     4921 aaccaagctg gagtgcagtg gcacaatctt ggctcactgc aatctccgcc tcctgggttc
     4981 aagcgattct cctgcctcag cctcccgagt tgttgggatt ccaggcatgc atgaccaggc
     5041 tcagctaatt tttgtttttt tggtagagac ggggtttcac catattggcc aggctggtct
     5101 ccaactccta atctcaggtg atctacccac cttggcctcc caaattgctg ggattacagg
     5161 cgtgaaccac tgctcccttc cctgtccttc tgattttgta ggtaaccacg tgcggaccga
     5221 gcgccggcgc gataagctat ctataacaag aaaatatata tataataagt tatcacgtaa
     5281 gtagaacatg aaataacaat ataattatcg tatgagttaa atcttaaaag tcacgtaaaa
     5341 gataatcatg cgtcattttg actcacgcgg tcgttatagt tcaaaatcag tgacacttac
     5401 cgcattgaca agcacgcctc acgggagctc caagcggcga ctgagatgtc ctaaatgcac
     5461 agcgacggat tcgcgctatt tagaaagaga gagcaatatt tcaagaatgc atgcgtcaat
     5521 tttacgcaga ctatctttct agggttaaga gctcctgaga tgcaggtaca tcccactgat
     5581 gagtcccaaa taggacgaaa cgcgcttcgg tgcgtctggg attccactgc tatccacgcg
     5641 gccgcacggc attacgttag acgtctaggc cacaccaccc tgaaggcgcc tgctcgcctc
     5701 tgatctgttg aagctaagca gggtcggtcc tggttagtac ttggatggga ctccgcctgg
     5761 taatagccgg taccgtaggc ttgcatggtc atagctgttt ccttgcgctg gggatgcggt
     5821 gggctctata gagagtcgat cgtgtgatcg aaggaacccc tagtgatgga gttggccact
     5881 ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg
     5941 ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gcatgtagag actgggcctc
     6001 atgggccttc cgcctatcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     6061 ttaacatggt catagctgtt tccttgcgta ttgggcgctc tccgcttcct cgctcactga
     6121 ctcgctgcgc tcggtcgttc gggtaaagcc tggggtgcct aaataaaacg aaaggcccag
     6181 tcttccgact gagcctttcg ttttattgag caaaaggcca gcaaaaggcc aggaaccgta
     6241 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     6301 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     6361 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     6421 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     6481 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     6541 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     6601 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     6661 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     6721 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     6781 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     6841 aaggatctca a
//