LOCUS       sequence-140738-        4896 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  an alpha-crystallin::Venus (green lens) reporter cassette flanked by
            FRT sites and with a single loxP site at the 3' end was inserted in 
            the middle of pKHR4 mcs. Genbank accession number is KU144823..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4896)
  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 4896)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.devcel.2016.02"; journalName: "Dev Cell"; date: 
            "2016-03-21- 21"; volume: "36"; issue: "6"; pages: "654-67"
COMMENT     Sequence Label: pKHR5
FEATURES             Location/Qualifiers
     source          1..4896
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(33..461)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(143..164)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     355..374
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     588..610
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     602..619
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     603..619
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     726..742
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     726..742
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    762..795
                     /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    859..993
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(896..915)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     950..969
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    1030..1237
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1226..1243
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(join(1261..1974,1975..1977,1978..1980))
                     /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(1306..1327)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1655..1674
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1914..1935
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    2546..2579
                     /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    complement(2581..2614)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(2629..2645)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2630..2646)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(2711..2729)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2750..2766)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2750..2766)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2763..2785)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2774..2790
                     /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(join(2798..2804,2805..2822,2823..2828))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2843..2864
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2981..2998)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3152..3740)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3232..3251)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3911..4702,4703..4771))
                     /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     4534..4553
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4772..4876)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
       61 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
      121 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
      181 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
      241 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
      301 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
      361 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
      421 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct
      481 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
      541 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
      601 ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tggagctcca
      661 ccgcggtggc ggccaggtcc tttagtcggg tattcgggta gggataacag ggtaatggcg
      721 gccgctctag aactagtgga tcccccgggc tgcaggaatt cgaagttcct attctctaga
      781 aagtatagga acttcttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg
      841 aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat
      901 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
      961 aaactcatca atgtatctta tcatgtctgg atcatcatcg atggtaccca ccgcggtggc
     1021 ggcgatctct ccccagcatg cctgctattg tcttcccaat cctccccctt gctgtcctgc
     1081 cccaccccac cccccagaat agaatgacac ctactcagac aatgcgatgc aatttcctca
     1141 ttttattagg aaaggacagt gggagtggca ccttccaggg tcaaggaagg cacgggggag
     1201 gggcaaacaa cagatggctg gcaactagaa ggcacagtcg aggctgatca gcgggccgct
     1261 ttacttgtac agctcgtcca tgccgagagt gatcccggcg gcggtcacga actccagcag
     1321 gaccatgtga tcgcgcttct cgttggggtc tttgctcagg gcggactggt agctcaggta
     1381 gtggttgtcg ggcagcagca cggggccgtc gccgatgggg gtgttctgct ggtagtggtc
     1441 ggcgagctgc acgccgccgt cctcgatgtt gtggcggatc ttgaagttgg ccttgatgcc
     1501 gttcttctgc ttgtcggcgg tgatatagac gttgtggctg ttgtagttgt actccagctt
     1561 gtgccccagg atgttgccgt cctccttgaa gtcgatgccc ttcagctcga tgcggttcac
     1621 cagggtgtcg ccctcgaact tcacctcggc gcgggtcttg tagttgccgt cgtccttgaa
     1681 gaagatggtg cgctcctgga cgtagccttc gggcatggcg gacttgaaga agtcgtgctg
     1741 cttcatgtgg tcggggtagc gggcgaagca ctgcaggccg tagcccaggg tggtcacgag
     1801 ggtgggccag ggcacgggca gcttgccggt ggtgcagatc agcttcaggg tcagcttgcc
     1861 gtaggtggca tcgccctcgc cctcgccgga cacgctgaac ttgtggccgt ttacgtcgcc
     1921 gtccagctcg accaggatgg gcaccacccc ggtgaacagc tcctcgccct tgctcaccat
     1981 gttgtcagac ctggtaactc cttactgtaa cttattgcac ctgatgaaca aaagatgtga
     2041 actgaacgca agtaggaggc taatagaggc tagtacagta caaagatccc caaatgatgt
     2101 gtgccggcct tagtggaggc agacccctgg ttatatacct gtgctgcaga aagaggggct
     2161 tacaaatgga tctctctgga atggcattat ctcatggtgg gaacagtgtt gtcagcagaa
     2221 tcagcggggt ggactctgac ctgagaatga cagtctagca ctgaatggct cagacaatac
     2281 tcttgtggcc aactatcacc ggaaaataag tgcctgccaa aacaaaccag accagatgaa
     2341 gctacatggc tgtagaacgt cagtatttaa tgtgttgtgg ccacttggtt caccagattt
     2401 aagagctaat ttttttaaat atcacagata atcaatcgct tttttttttt cttgactgaa
     2461 aagcttagac tggaaaattc tttgaaaaag tttgcactac agaaaataaa tacataacat
     2521 cctgcactga atgcacacta ttaatgaagt tcctattctc tagaaagtat aggaacttca
     2581 ataacttcgt atagcataca ttatacgaag ttatgatatc aagcttatcg ataccgtcga
     2641 cctcgagccg ccattaccct gttatcccta cccgaatacc cgactaaagg accggtacca
     2701 gcttttgttc cctttagtga gggttaattt cgagcttggc gtaatcatgg tcatagctgt
     2761 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     2821 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     2881 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     2941 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     3001 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     3061 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     3121 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     3181 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     3241 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     3301 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     3361 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     3421 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     3481 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     3541 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat
     3601 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     3661 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     3721 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     3781 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     3841 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     3901 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     3961 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     4021 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     4081 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     4141 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     4201 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     4261 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     4321 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     4381 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     4441 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     4501 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     4561 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     4621 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     4681 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     4741 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     4801 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     4861 aaataggggt tccgcgcaca tttccccgaa aagtgc
//
