LOCUS       40924_47008        8509 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector px330_DBH-p2a-FLPo, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8509)
  AUTHORS   Sun JJ, Ray R.
  TITLE     Generation of Two Noradrenergic-Specific 
            Dopamine-Beta-Hydroxylase-FLPo Knock-In Mice Using 
            CRISPR/Cas9-Mediated Targeting in Embryonic Stem Cells
  JOURNAL   PLoS ONE 11 (7), E0159474 (2016)
  PUBMED    27441631
REFERENCE   2  (bases 1 to 8509)
  AUTHORS   Sun JJ, Ray R.
  TITLE     Direct Submission
  JOURNAL   Submitted (27-APR-2016) Neuroscience, Baylor College of Medicine, 
            One Baylor Plaza Room T707, Houston, TX 77030, USA
REFERENCE   3  (bases 1 to 8509)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8509)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "PLoS ONE"; 
            date: "2016"; volume: "11"; issue: "7"; pages: "E0159474"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (27-APR-2016) Neuroscience, Baylor College of Medicine, One Baylor 
            Plaza Room T707, Houston, TX 77030, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8509
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1..270
                     /label=CAG
                     /note="CAG"
                     /regulatory_class="enhancer"
     promoter        284..561
                     /label=chicken beta-actin promoter
     intron          562..790
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      802..811
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             811..876
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             883..903
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             928..5028
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     CDS             5029..5076
                     /codon_start=1
                     /product="bipartite nuclear localization signal from
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     polyA_signal    5110..5317
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5326..5466
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      5541..5996
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        6278..6382
                     /label=AmpR promoter
     CDS             6383..7240
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      7414..8002
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     promoter        8064..8304
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_feature    8314..8333
                     /label=DBH-p2a-FLPo sgRNA
                     /note="DBH-p2a-FLPo sgRNA"
     misc_RNA        8334..8409
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        8506..8509
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer;
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
       61 tcaatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
      121 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
      181 aatgacggta aatggcccgc ctggcattgt gcccagtaca tgaccttatg ggactttcct
      241 acttggcagt acatctacgt attagtcatc gctattacca tggtcgaggt gagccccacg
      301 ttctgcttca ctctccccat ctcccccccc tccccacccc caattttgta tttatttatt
      361 ttttaattat tttgtgcagc gatgggggcg gggggggggg gggggcgcgc gccaggcggg
      421 gcggggcggg gcgaggggcg gggcggggcg aggcggagag gtgcggcggc agccaatcag
      481 agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg gccctataaa
      541 aagcgaagcg cgcggcgggc gggagtcgct gcgacgctgc cttcgccccg tgccccgctc
      601 cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag
      661 cgggcgggac ggcccttctc ctccgggctg taattagctg agcaagaggt aagggtttaa
      721 gggatggttg gttggtgggg tattaatgtt taattacctg gagcacctgc ctgaaatcac
      781 tttttttcag gttggaccgg tgccaccatg gactataagg accacgacgg agactacaag
      841 gatcatgata ttgattacaa agacgatgac gataagatgg ccccaaagaa gaagcggaag
      901 gtcggtatcc acggagtccc agcagccgac aagaagtaca gcatcggcct ggacatcggc
      961 accaactctg tgggctgggc cgtgatcacc gacgagtaca aggtgcccag caagaaattc
     1021 aaggtgctgg gcaacaccga ccggcacagc atcaagaaga acctgatcgg agccctgctg
     1081 ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga gaaccgccag aagaagatac
     1141 accagacgga agaaccggat ctgctatctg caagagatct tcagcaacga gatggccaag
     1201 gtggacgaca gcttcttcca cagactggaa gagtccttcc tggtggaaga ggataagaag
     1261 cacgagcggc accccatctt cggcaacatc gtggacgagg tggcctacca cgagaagtac
     1321 cccaccatct accacctgag aaagaaactg gtggacagca ccgacaaggc cgacctgcgg
     1381 ctgatctatc tggccctggc ccacatgatc aagttccggg gccacttcct gatcgagggc
     1441 gacctgaacc ccgacaacag cgacgtggac aagctgttca tccagctggt gcagacctac
     1501 aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa ggccatcctg
     1561 tctgccagac tgagcaagag cagacggctg gaaaatctga tcgcccagct gcccggcgag
     1621 aagaagaatg gcctgttcgg aaacctgatt gccctgagcc tgggcctgac ccccaacttc
     1681 aagagcaact tcgacctggc cgaggatgcc aaactgcagc tgagcaagga cacctacgac
     1741 gacgacctgg acaacctgct ggcccagatc ggcgaccagt acgccgacct gtttctggcc
     1801 gccaagaacc tgtccgacgc catcctgctg agcgacatcc tgagagtgaa caccgagatc
     1861 accaaggccc ccctgagcgc ctctatgatc aagagatacg acgagcacca ccaggacctg
     1921 accctgctga aagctctcgt gcggcagcag ctgcctgaga agtacaaaga gattttcttc
     1981 gaccagagca agaacggcta cgccggctac attgacggcg gagccagcca ggaagagttc
     2041 tacaagttca tcaagcccat cctggaaaag atggacggca ccgaggaact gctcgtgaag
     2101 ctgaacagag aggacctgct gcggaagcag cggaccttcg acaacggcag catcccccac
     2161 cagatccacc tgggagagct gcacgccatt ctgcggcggc aggaagattt ttacccattc
     2221 ctgaaggaca accgggaaaa gatcgagaag atcctgacct tccgcatccc ctactacgtg
     2281 ggccctctgg ccaggggaaa cagcagattc gcctggatga ccagaaagag cgaggaaacc
     2341 atcaccccct ggaacttcga ggaagtggtg gacaagggcg cttccgccca gagcttcatc
     2401 gagcggatga ccaacttcga taagaacctg cccaacgaga aggtgctgcc caagcacagc
     2461 ctgctgtacg agtacttcac cgtgtataac gagctgacca aagtgaaata cgtgaccgag
     2521 ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa aggccatcgt ggacctgctg
     2581 ttcaagacca accggaaagt gaccgtgaag cagctgaaag aggactactt caagaaaatc
     2641 gagtgcttcg actccgtgga aatctccggc gtggaagatc ggttcaacgc ctccctgggc
     2701 acataccacg atctgctgaa aattatcaag gacaaggact tcctggacaa tgaggaaaac
     2761 gaggacattc tggaagatat cgtgctgacc ctgacactgt ttgaggacag agagatgatc
     2821 gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag
     2881 cggcggagat acaccggctg gggcaggctg agccggaagc tgatcaacgg catccgggac
     2941 aagcagtccg gcaagacaat cctggatttc ctgaagtccg acggcttcgc caacagaaac
     3001 ttcatgcagc tgatccacga cgacagcctg acctttaaag aggacatcca gaaagcccag
     3061 gtgtccggcc agggcgatag cctgcacgag cacattgcca atctggccgg cagccccgcc
     3121 attaagaagg gcatcctgca gacagtgaag gtggtggacg agctcgtgaa agtgatgggc
     3181 cggcacaagc ccgagaacat cgtgatcgaa atggccagag agaaccagac cacccagaag
     3241 ggacagaaga acagccgcga gagaatgaag cggatcgaag agggcatcaa agagctgggc
     3301 agccagatcc tgaaagaaca ccccgtggaa aacacccagc tgcagaacga gaagctgtac
     3361 ctgtactacc tgcagaatgg gcgggatatg tacgtggacc aggaactgga catcaaccgg
     3421 ctgtccgact acgatgtgga ccatatcgtg cctcagagct ttctgaagga cgactccatc
     3481 gacaacaagg tgctgaccag aagcgacaag aaccggggca agagcgacaa cgtgccctcc
     3541 gaagaggtcg tgaagaagat gaagaactac tggcggcagc tgctgaacgc caagctgatt
     3601 acccagagaa agttcgacaa tctgaccaag gccgagagag gcggcctgag cgaactggat
     3661 aaggccggct tcatcaagag acagctggtg gaaacccggc agatcacaaa gcacgtggca
     3721 cagatcctgg actcccggat gaacactaag tacgacgaga atgacaagct gatccgggaa
     3781 gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt tccggaagga tttccagttt
     3841 tacaaagtgc gcgagatcaa caactaccac cacgcccacg acgcctacct gaacgccgtc
     3901 gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt gtacggcgac
     3961 tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg agcaggaaat cggcaaggct
     4021 accgccaagt acttcttcta cagcaacatc atgaactttt tcaagaccga gattaccctg
     4081 gccaacggcg agatccggaa gcggcctctg atcgagacaa acggcgaaac cggggagatc
     4141 gtgtgggata agggccggga ttttgccacc gtgcggaaag tgctgagcat gccccaagtg
     4201 aatatcgtga aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc
     4261 aagaggaaca gcgataagct gatcgccaga aagaaggact gggaccctaa gaagtacggc
     4321 ggcttcgaca gccccaccgt ggcctattct gtgctggtgg tggccaaagt ggaaaagggc
     4381 aagtccaaga aactgaagag tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc
     4441 agcttcgaga agaatcccat cgactttctg gaagccaagg gctacaaaga agtgaaaaag
     4501 gacctgatca tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg ccggaagaga
     4561 atgctggcct ctgccggcga actgcagaag ggaaacgaac tggccctgcc ctccaaatat
     4621 gtgaacttcc tgtacctggc cagccactat gagaagctga agggctcccc cgaggataat
     4681 gagcagaaac agctgtttgt ggaacagcac aagcactacc tggacgagat catcgagcag
     4741 atcagcgagt tctccaagag agtgatcctg gccgacgcta atctggacaa agtgctgtcc
     4801 gcctacaaca agcaccggga taagcccatc agagagcagg ccgagaatat catccacctg
     4861 tttaccctga ccaatctggg agcccctgcc gccttcaagt actttgacac caccatcgac
     4921 cggaagaggt acaccagcac caaagaggtg ctggacgcca ccctgatcca ccagagcatc
     4981 accggcctgt acgagacacg gatcgacctg tctcagctgg gaggcgacaa aaggccggcg
     5041 gccacgaaaa aggccggcca ggcaaaaaag aaaaagtaag aattcctaga gctcgctgat
     5101 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     5161 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     5221 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     5281 gggaggattg ggaagagaat agcaggcatg ctggggagcg gccgcaggaa cccctagtga
     5341 tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg
     5401 tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc
     5461 tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     5521 tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     5581 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     5641 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     5701 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg
     5761 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     5821 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     5881 gggctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     5941 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg
     6001 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     6061 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     6121 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     6181 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     6241 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     6301 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     6361 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     6421 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     6481 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     6541 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     6601 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     6661 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     6721 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     6781 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     6841 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     6901 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     6961 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     7021 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     7081 tggagccggt gagcgtggaa gccgcggtat cattgcagca ctggggccag atggtaagcc
     7141 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     7201 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     7261 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     7321 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     7381 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     7441 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     7501 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     7561 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     7621 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     7681 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     7741 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     7801 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     7861 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     7921 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     7981 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     8041 ttgctggcct tttgctcaca tgtgagggcc tatttcccat gattccttca tatttgcata
     8101 tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat
     8161 tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat
     8221 tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg
     8281 ctttatatat cttgtggaaa ggacgaaaca ccgagaatag cttctcacaa ggtgttttag
     8341 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
     8401 agtcggtgct tttttgtttt agagctagaa atagcaagtt aaaataaggc tagtccgttt
     8461 ttagcgcgtg cgccaattct gcagacaaat ggctctagag gtacccgtt
//
