LOCUS       40924_575        7156 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector DBH-p2a-FLPo, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7156)
  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 7156)
  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 7156)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7156)
  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..7156
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      18..562
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin."
     misc_feature    836..1835
                     /label=5' homology arm
                     /note="5' homology arm"
     CDS             1845..1901
                     /codon_start=1
                     /label=P2A
                     /note="2A peptide from porcine teschovirus-1 polyprotein"
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1902..3197
                     /codon_start=1
                     /label=FLPo
                     /note="nuclear-targeted site-specific recombinase"
                     /translation="MAPKKKRKVMSQFDILCKTPPKVLVRQFVERFERPSGEKIASCAA
                     ELTYLCWMITHNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKK
                     LIPAWEFTIIPYNGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESI
                     WEITEKILNSFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGV
                     IIQCLVTETKTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEY
                     QLLKDNLVRSYNKALKKNAPYPIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWS
                     DKRASAVARTTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQL
                     KGSAEGSIRYPAWNGIISQEVLDYLSSYINRRI"
     protein_bind    3201..3234
                     /label=lox2272
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     promoter        3241..3740
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     promoter        3752..3799
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter"
     CDS             3818..4618
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase from Tn5"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    4659..4883
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    4938..4971
                     /label=Lox2722
                     /note="Lox2722"
     protein_bind    complement(4938..4971)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites."
     misc_feature    4972..5968
                     /label=3' homology arm with PAM motif deleted
                     /note="3' homology arm with PAM motif deleted"
     promoter        6044..6148
                     /label=AmpR promoter
     CDS             6149..7006
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 ttaataagat gatcttcttg agatcgtttt ggtctgcgcg taatctcttg ctctgaaaac
       61 gaaaaaaccg ccttgcaggg cggtttttcg aaggttctct gagctaccaa ctctttgaac
      121 cgaggtaact ggcttggagg agcgcagtca ccaaaacttg tcctttcagt ttagccttaa
      181 ccggcgcatg acttcaagac taactcctct aaatcaatta ccagtggctg ctgccagtgg
      241 tgcttttgca tgtctttccg ggttggactc aagacgatag ttaccggata aggcgcagcg
      301 gtcggactga acggggggtt cgtgcataca gtccagcttg gagcgaactg cctacccgga
      361 actgagtgtc aggcgtggaa tgagacaaac gcggccataa cagcggaatg acaccggtaa
      421 accgaaaggc aggaacagga gagcgcacga gggagccgcc aggggaaacg cctggtatct
      481 ttatagtcct gtcgggtttc gccaccactg atttgagcgt cagatttcgt gatgcttgtc
      541 aggggggcgg agcctatgga aaaacggctt tgccgcggcc ctctcacttc cctgttaagt
      601 atcttcctgg catcttccag gaaatctccg ccccgttcgt aagccatttc cgctcgccgc
      661 agtcgaacga ccgagcgtag cgagtcagtg agcgaggaag cggaatatat cctgtatcac
      721 atattctgct gacgcaccgg tgcagccttt tttctcctgc cacatgaagc acttcactga
      781 caccctcatc agtgccaaca tagtaagcca gtatacactc cgctagcgaa ttgatcaagc
      841 agtgcttaca tgagactgag aaaatgacca cacagtgata gagacaggcg tacagacaga
      901 tagatggtaa gggaacaggc cagcaacact gccaggaagg ccagagaggc cacttctcct
      961 gggcatagaa caacagagga ctaacctcag ccctgtctgg atggtgtgtg actgagtgtc
     1021 agcaccctgc tcagagaata aaaatagagc tatgggcagg ggaggcaaga aaccagcagt
     1081 ctatgacaag agccccacag cgcagcttct ccctccttat ctgaggcctg ctgggattca
     1141 aaggcaccta atttatttcc cagtagagct agccagcagc agcagtgcca gcccagagtg
     1201 ctggtcagca gaccccgcct gctgtgttag gcaggggagg caggcactgc agcagaaggt
     1261 gaacagacct ttcctggcag cgtttgggta ccggattaat tgctaaccca aacacccatg
     1321 ccatggggag acttgttcat cccagagcag tttgaggcac cgccggatat ggttagatct
     1381 tggcttttgc agacgccttg ttggacagtg gctaaaccca cggcctgcct ccaaagactc
     1441 tgatcctttc ctcggagtat acagttgagt gcaggcatgg aggtggagac gtctggaggg
     1501 cttcaggttg ctgacagaga acacaacagc ctagggaagg gacgtggatc ctccggtgct
     1561 aactctcaaa ggctgacttg gaagaggtgt ggagtgttca gcatgggaga aatgatttta
     1621 aggacaagaa tccaaccagt tagggagata gcagtttgct tggcccctcc agcctcggct
     1681 taccttctgc tccttcctta cagggtgagt ggaatctgca gcctttgcct aagatcacct
     1741 ccacgctgga agaacccacc ccacgctgcc ccatccgaca gactcagagc cccgctaacc
     1801 ccactgtgcc catcacaaca gaggcagatg ctgaaggcag cggcgccacg aacttctctc
     1861 tgttaaagca agcaggagac gtggaagaaa accccggtcc tatggctcct aagaagaaga
     1921 ggaaggtgat gagccagttc gacatcctgt gcaagacccc ccccaaggtg ctggtgcggc
     1981 agttcgtgga gagattcgag aggcccagcg gcgagaagat cgccagctgt gccgccgagc
     2041 tgacctacct gtgctggatg atcacccaca acggcaccgc catcaagagg gccaccttca
     2101 tgagctacaa caccatcatc agcaacagcc tgagcttcga catcgtgaac aagagcctgc
     2161 agttcaagta caagacccag aaggccacca tcctggaggc cagcctgaag aagctgatcc
     2221 ccgcctggga gttcaccatc atcccttaca acggccagaa gcaccagagc gacatcaccg
     2281 acatcgtgtc cagcctgcag ctgcagttcg agagcagcga ggaggccgac aagggcaaca
     2341 gccacagcaa gaagatgctg aaggccctgc tgtccgaggg cgagagcatc tgggagatca
     2401 ccgagaagat cctgaacagc ttcgagtaca ccagcaggtt caccaagacc aagaccctgt
     2461 accagttcct gttcctggcc acattcatca actgcggcag gttcagcgac atcaagaacg
     2521 tggaccccaa gagcttcaag ctggtgcaga acaagtacct gggcgtgatc attcagtgcc
     2581 tggtgaccga gaccaagaca agcgtgtcca ggcacatcta ctttttcagc gccagaggca
     2641 ggatcgaccc cctggtgtac ctggacgagt tcctgaggaa cagcgagccc gtgctgaaga
     2701 gagtgaacag gaccggcaac agcagcagca acaagcagga gtaccagctg ctgaaggaca
     2761 acctggtgcg cagctacaac aaggccctga agaagaacgc cccctacccc atcttcgcta
     2821 tcaagaacgg ccctaagagc cacatcggca ggcacctgat gaccagcttt ctgagcatga
     2881 agggcctgac cgagctgaca aacgtggtgg gcaactggag cgacaagagg gcctccgccg
     2941 tggccaggac cacctacacc caccagatca ccgccatccc cgaccactac ttcgccctgg
     3001 tgtccaggta ctacgcctac gaccccatca gcaaggagat gatcgccctg aaggacgaga
     3061 ccaaccccat cgaggagtgg cagcacatcg agcagctgaa gggcagcgcc gagggcagca
     3121 tcagataccc cgcctggaac ggcatcatca gccaggaggt gctggactac ctgagcagct
     3181 acatcaacag gcggatctag ataacttcgt ataaagtatc ctatacgaag ttattctacc
     3241 gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca gccccgctgg
     3301 gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc accggtaggc
     3361 gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct cccctagtca
     3421 ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac
     3481 gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg gtaggccttt
     3541 ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg ctgggaaggg
     3601 gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct
     3661 ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt
     3721 cctcatctcc gggcctttcg acctgcagcc tgttgacaat taatcatcgg catagtatat
     3781 cggcatagta taatacgaca aggtgaggaa ctaaaccatg ggatcggcca ttgaacaaga
     3841 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     3901 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     3961 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc
     4021 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     4081 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     4141 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     4201 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     4261 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     4321 cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg atgatctcgt
     4381 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     4441 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     4501 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     4561 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     4621 aggggatcaa ttctctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc
     4681 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     4741 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     4801 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     4861 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctcgactag
     4921 agcttgcgga acccttaata acttcgtata aagtatccta tacgaagtta taaccttgtg
     4981 agaagctatt cttcagaccc tcctcctcct gtccacctgg gctcacacca gctctgtgta
     5041 gccctccctt cctgtgtgaa gactcacttc catagactag tgctggccat ccagggagaa
     5101 ggagcatgtc accttggaga cacttgcatg actgcacccc atgaggtcca actgaacgga
     5161 gaagccctgg acattatcta cacactgctg actccacata gggacagctc gctgtggtag
     5221 tccagattca acaccacctt gcctccagaa cagccttgcc aggctgggtg cagactcccc
     5281 aagccccaca gtcccgaccc tgttggtcct ctggtggtga cttgtgttag tgtatgccgt
     5341 gacaacactg cttaaatatt cctttgcaga gtggctcatg tttcccagtg ggcggcttcc
     5401 ctgcaacaag acaggacaag tcgtttagca gttagagact caccagggga actcgcttcg
     5461 tggcagagta aatagatatt ttcgcccacc tagaggaaac ccccaacaag tagttccatg
     5521 acggagagcc aagatggcta gagccaggct ctgtagagga gtggaacggc cagagctggt
     5581 tgcttctcct gatggtgaca ggccaggctc tgaaatagat agttaggtga agagctggct
     5641 tgggccctta atgaaggagt gactagagca agccactcag ctgcctgatc ttcagtctcc
     5701 ttgtctgtta aatgagactt acccttacct agcactaact gtgtacacat atgcatgtcc
     5761 aggattcctg ccctgtgctg agcctttctg ggctctatga tatcagacat gttgtcccat
     5821 gtgagtgagg cctgcttcta taattctagt tacttctacc ctccctgact tcttcccctc
     5881 agtctcctgc tgaagggcca gctcagctct ttgatggttt tggaagggag taggagaccg
     5941 ggccttttca gctatttcag caaaatgcat catttaaata atttctagaa agcttaattc
     6001 gtaccgatct aacgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     6061 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     6121 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     6181 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     6241 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     6301 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     6361 agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg
     6421 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     6481 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     6541 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     6601 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     6661 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact
     6721 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     6781 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     6841 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     6901 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     6961 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     7021 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     7081 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     7141 ctgagcgtca gacccc
//
