LOCUS       Exported                6737 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  a plasmid derived from pKHR5: in addition to Venus reporter gene, it
            also contains cmlc2::mCherry (red heart) reporter gene at 3' end of 
            the mcs as pKHR7. Genbank accession number is KU144825..
ACCESSION   .
VERSION     .
KEYWORDS    pKHR8
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6737)
  AUTHORS   Hoshijima K, Jurynec MJ, Grunwald DJ
  TITLE     Precise Editing of the Zebrafish Genome Made Simple and Efficient.
  JOURNAL   Dev Cell. 2016 Mar 21;36(6):654-67. doi: 
            10.1016/j.devcel.2016.02.015.
  PUBMED    27003937
REFERENCE   2  (bases 1 to 6737)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6737
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     178..197
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     437..458
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    1069..1102
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     protein_bind    1104..1137
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     primer_bind     complement(1152..1168)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1153..1169)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     CDS             2063..2773
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEDDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2208..2226)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2476..2495
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2670..2693
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     promoter        complement(2789..2806)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    2811..2945
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(3075..3093)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3114..3130)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3114..3130)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3127..3149)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3138..3154
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(3162..3192)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3207..3228
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3345..3362)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3516..4104)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3596..3615)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4275..5135)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4898..4917
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5136..5240)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5266..5721)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5403..5424)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5615..5634
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5848..5870
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5862..5879
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5863..5879
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5886..5905
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5886..5904
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     5986..6002
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5986..6002
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    6022..6055
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     polyA_signal    6119..6253
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    6290..6497
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     6486..6503
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(6521..503)
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6566..6587)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 atcttgaagt tggccttgat gccgttcttc tgcttgtcgg cggtgatata gacgttgtgg
       61 ctgttgtagt tgtactccag cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg
      121 cccttcagct cgatgcggtt caccagggtg tcgccctcga acttcacctc ggcgcgggtc
      181 ttgtagttgc cgtcgtcctt gaagaagatg gtgcgctcct ggacgtagcc ttcgggcatg
      241 gcggacttga agaagtcgtg ctgcttcatg tggtcggggt agcgggcgaa gcactgcagg
      301 ccgtagccca gggtggtcac gagggtgggc cagggcacgg gcagcttgcc ggtggtgcag
      361 atcagcttca gggtcagctt gccgtaggtg gcatcgccct cgccctcgcc ggacacgctg
      421 aacttgtggc cgtttacgtc gccgtccagc tcgaccagga tgggcaccac cccggtgaac
      481 agctcctcgc ccttgctcac catgttgtca gacctggtaa ctccttactg taacttattg
      541 cacctgatga acaaaagatg tgaactgaac gcaagtagga ggctaataga ggctagtaca
      601 gtacaaagat ccccaaatga tgtgtgccgg ccttagtgga ggcagacccc tggttatata
      661 cctgtgctgc agaaagaggg gcttacaaat ggatctctct ggaatggcat tatctcatgg
      721 tgggaacagt gttgtcagca gaatcagcgg ggtggactct gacctgagaa tgacagtcta
      781 gcactgaatg gctcagacaa tactcttgtg gccaactatc accggaaaat aagtgcctgc
      841 caaaacaaac cagaccagat gaagctacat ggctgtagaa cgtcagtatt taatgtgttg
      901 tggccacttg gttcaccaga tttaagagct aattttttta aatatcacag ataatcaatc
      961 gctttttttt tttcttgact gaaaagctta gactggaaaa ttctttgaaa aagtttgcac
     1021 tacagaaaat aaatacataa catcctgcac tgaatgcaca ctattaatga agttcctatt
     1081 ctctagaaag tataggaact tcaataactt cgtatagcat acattatacg aagttatgat
     1141 atcaagctta tcgataccgt cgacctcgag aaatcagttg tgttaaataa gagacattca
     1201 aaataaatgt aaatgagctc tccaaatcag cagacttaac attctttaaa atgattgatt
     1261 caatagtgat aaaaatcagg catagccagt tgtaacttta gataaattac agaaaatgtc
     1321 aaatacagag aaccgattct tttttatgat acatccaagc acacatttaa cacaatccag
     1381 gcaaaccccg aatttcacag tcacaagcac tgtttgtaca agagctttgc ctaaggacac
     1441 acagtctcta taagtccagg tcgttggttt cactcttatt ttaaacatgt gacatttttc
     1501 ctgccatcct gtcttaggct gctgtttgct tcattccatg tcacattaaa ttcctcagta
     1561 gcacctttta cacacacagc caatcttttc cagaaaattc aattgctttg aagagataat
     1621 gtgtgaacaa atccatttag aaaaggaaaa ttaagaattt gtaaaatcat ctgtaaattg
     1681 ttggcattct tctgtatatg aacatcacat catttacagg taaaggtctg gtcattaatt
     1741 atatgacaat ttactggtat tattttgtga aaggggctat tttcaatgcg ttcatccatc
     1801 cttttcatcc ctcaaatctc tcattcacgt ccccctcccc atctgcacac tttatctcat
     1861 tttccaccct gctggaatct gagcacttgt gcagttatca gggctcctgt atttaggagg
     1921 ctctgggtgt ccatgtaggg gacgaacaga aacactgcag acctttatag aagaacaatt
     1981 gataagagtc ctcatacata aagactccat tagtaagcca gtgacccagg agcccagacc
     2041 aacagcaaag cagacagtga ccatggtgag caagggcgag gacgacaaca tggccatcat
     2101 caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga
     2161 gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt
     2221 gaccaagggc ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg
     2281 ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc
     2341 cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac
     2401 ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa
     2461 cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg cctcctccga
     2521 gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa
     2581 ggacggcggc cactacgacg ccgaggtcaa gaccacctac aaggccaaga agcccgtgca
     2641 gctgcccggc gcctacaacg tcaacatcaa gctggacatc acctcccaca acgaggacta
     2701 caccatcgtg gaacagtacg agcgcgccga gggccgccac tccaccggcg gcatggacga
     2761 gctgtacaag taaggcgcgc ctctagaact atagtgagtc gtattacgta gatccagaca
     2821 tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct
     2881 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac
     2941 aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg
     3001 tttttttcga gccgccatta ccctgttatc cctacccgaa tacccgacta aaggaccggt
     3061 accagctttt gttcccttta gtgagggtta atttcgagct tggcgtaatc atggtcatag
     3121 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     3181 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     3241 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     3301 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     3361 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     3421 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     3481 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     3541 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     3601 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     3661 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     3721 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     3781 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     3841 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     3901 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     3961 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     4021 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     4081 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     4141 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     4201 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     4261 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     4321 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     4381 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     4441 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     4501 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     4561 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     4621 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     4681 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     4741 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     4801 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     4861 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     4921 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     4981 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     5041 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     5101 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     5161 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     5221 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta
     5281 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     5341 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     5401 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     5461 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     5521 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     5581 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     5641 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     5701 atgcgccgct acagggcgcg tcccattcgc cattcaggct gcgcaactgt tgggaagggc
     5761 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc
     5821 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg
     5881 aattgtaata cgactcacta tagggcgaat tggagctcca ccgcggtggc ggccaggtcc
     5941 tttagtcggg tattcgggta gggataacag ggtaatggcg gccgctctag aactagtgga
     6001 tcccccgggc tgcaggaatt cgaagttcct attctctaga aagtatagga acttcttaaa
     6061 aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa
     6121 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     6181 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     6241 tcatgtctgg atcatcatcg atggtaccca ccgcggtggc ggcgatctct ccccagcatg
     6301 cctgctattg tcttcccaat cctccccctt gctgtcctgc cccaccccac cccccagaat
     6361 agaatgacac ctactcagac aatgcgatgc aatttcctca ttttattagg aaaggacagt
     6421 gggagtggca ccttccaggg tcaaggaagg cacgggggag gggcaaacaa cagatggctg
     6481 gcaactagaa ggcacagtcg aggctgatca gcgggccgct ttacttgtac agctcgtcca
     6541 tgccgagagt gatcccggcg gcggtcacga actccagcag gaccatgtga tcgcgcttct
     6601 cgttggggtc tttgctcagg gcggactggt agctcaggta gtggttgtcg ggcagcagca
     6661 cggggccgtc gccgatgggg gtgttctgct ggtagtggtc ggcgagctgc acgccgccgt
     6721 cctcgatgtt gtggcgg
//
