LOCUS       sequence-361365-        4081 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient expression of humanized KO1 in plant cell.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4081)
  AUTHORS   Fujii Y, Yoshimura A, Kodama Y
  TITLE     A novel orange-colored bimolecular fluorescence complementation 
            (BiFC) assay using monomeric Kusabira-Orange protein.
  JOURNAL   Biotechniques. 2018 Apr;64(4):153-161. doi: 10.2144/btn-2017-0121.
  PUBMED    29661017
REFERENCE   2  (bases 1 to 4081)
  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"; journalName: 
            "Biotechniques"; date: "2018-04"; volume: "64"; issue: "4"; pages: 
            "153-161"
COMMENT     Sequence Label: p35S-hKO1
FEATURES             Location/Qualifiers
     source          1..4081
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     213..232
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     466..483
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    600..621
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(636..641,642..659,660..666)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    674..690
                     /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)."
     primer_bind     679..701
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     698..714
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     698..714
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     terminator      complement(752..1004)
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     CDS             complement(1023..1679)
                     /codon_start=1
                     /product="humanized Kusabira-Orange fluorescent protein"
                     /label=hKO
                     /note="mammalian codon-optimized"
                     /translation="MVSVIKPEMKMKYFMDGSVNGHEFTVEGEGTGKPYEGHQEMTLRV
                     TMAKGGPMPFSFDLVSHTFCYGHRPFTKYPEEIPDYFKQAFPEGLSWERSLQFEDGGFA
                     AVSAHISLRGNCFEHKSKFVGVNFPADGPVMQNQSSDWEPSTEKITTCDGVLKGDVTMY
                     LKLAGGGNHKCQFKTTYKAAKKILKMPQSHFIGHRLVRKTEGNITELVEDAVAHC"
     misc_feature    1699..1754
                     /label=TMV Omega
                     /note="translational enhancer from the tobacco mosaic virus
                     5'-leader sequence (Gallie et al., 1988)"
     promoter        complement(1774..2119)
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     complement(1811..1831)
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     primer_bind     complement(2179..2196)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2179..2195)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2188..2210)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2404..2423)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2523..2545
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2583..2601)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2669..2773
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2774..2842,2843..3634)
                     /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     complement(2992..3011)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(3805..4081,1..312)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
       61 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
      121 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
      181 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
      241 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
      301 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
      361 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
      421 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
      481 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
      541 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
      601 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
      661 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgacatgatt
      721 acgaattctc atgtttgaca gcttatcatc ggatctagta acatagatga caccgcgcgc
      781 gataatttat cctagtttgc gcgctatatt ttgttttcta tcgcgtatta aatgtataat
      841 tgcgggactc taatcataaa aacccatctc ataaataacg tcatgcatta catgttaatt
      901 attacatgct taacgtaatt caacagaaat tatatgataa tcatcgcaag accggcaaca
      961 ggattcaatc ttaagaaact ttattgccaa atgtttgaac gatctgcagc cgggcggccg
     1021 ctttagcagt gggccacggc gtcctccacc agctcggtga tgttgccctc ggtcttcctc
     1081 accagcctgt ggccgatgaa gtggctctgg ggcatcttca ggatcttctt ggcggccttg
     1141 taggtggtct tgaactggca cttgtggttg ccgccgccgg ccagcttcag gtacatggtc
     1201 acgtcgccct tcagcacgcc gtcgcaggtg gtgatcttct cggtgctggg ctcccagtcg
     1261 ctgctctggt tctgcatcac ggggccgtcg gcggggaagt tcacgcccac gaacttgctc
     1321 ttgtgctcga agcagttgcc cctcaggctg atgtgggcgc tcacggcggc gaagccgccg
     1381 tcctcgaact ggaggctcct ctcccagctc aggccctcgg ggaaggcctg cttgaagtag
     1441 tcggggatct cctcggggta cttggtgaag ggcctgtggc cgtagcagaa ggtgtggctc
     1501 accaggtcga agctgaaggg catggggccg cccttggcca ttgtcaccct cagggtcatc
     1561 tcctggtggc cctcgtaggg cttgccggtg ccctcgccct ccacggtgaa ctcgtggccg
     1621 ttcacgctgc cgtccatgaa gtacttcatc ttcatctcgg gcttgatcac gctcaccatg
     1681 gatcctctag agtcgactgt aattgtaaat agtaattgta atgttgtttg ttgtttgttg
     1741 ttgttggtaa ttgttgaagg atctcaagaa gcctgtcctc tccaaatgaa atgaacttcc
     1801 ttatatagag gaagggtctt gcgaaggata gtgggattgt gcgtcatccc ttacgtcagt
     1861 ggagatatca catcaatcca cttgctttga agacgtggtt ggaacgtctt ctttttccac
     1921 gatgttcctc gtgggtgggg gtccatcttt gggaccactg tcggtagagg catcttgaac
     1981 gatagccttt cctttatcgc aatgatggca tttgtaggag ccaccttcct tttctactgt
     2041 cctttcgatg aagtgacaga tagctgggca atggaatccg aggaggtttc ccgatattac
     2101 cctttgttga aaagtctcaa tagccctctg gtcttctgag gggggatcct ctagagcctg
     2161 caggcatgca agcttggcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
     2221 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
     2281 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct
     2341 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct
     2401 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc
     2461 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     2521 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa
     2581 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     2641 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     2701 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     2761 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     2821 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     2881 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     2941 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     3001 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     3061 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     3121 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     3181 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     3241 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     3301 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     3361 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     3421 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     3481 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     3541 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     3601 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     3661 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     3721 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     3781 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     3841 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     3901 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     3961 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     4021 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     4081 c
//
