LOCUS       sequence-456430-        8833 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Efficient screening of gene editing in plants (kanamycin 
            resistance)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8833)
  AUTHORS   Kong D, Cheng Y, Ke Y, Dang X, Liu X, Wang C, Huang C, Ye R, Miki D
  TITLE     SSA-mediated selection marker gene activation enhances relative 
            gene-targeting efficiency in plants.
  JOURNAL   Horticulture Research, 06 Aug 2025, uhaf196
REFERENCE   2  (bases 1 to 8833)
  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"; journalName: 
            "Horticulture Research, 06 Aug 2025, uhaf196"
COMMENT     Sequence Label: pCambia1300-NP-PTII
FEATURES             Location/Qualifiers
     source          1..8833
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(60..77)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(60..76)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(69..91)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     misc_feature    279..303
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             1604..2233
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             2662..3735
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="MSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
                     QAAADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
                     KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
                     GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
                     ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
                     LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
                     LVMRYRNLIEGEASAGS"
     rep_origin      3801..3995
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(4231..4253)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4339..4479
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4394..4413
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4494..4511)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4665..5253)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4745..4764)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5340..6134)
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     misc_feature    6559..6583
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     polyA_signal    6661..6835
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     primer_bind     complement(7003..7022)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     7734..7753
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(7843..8519)
                     /label=CaMV 35S promoter (enhanced)
                     /note="cauliflower mosaic virus 35S promoter with a 
                     duplicated enhancer region"
     primer_bind     complement(7886..7906)
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     protein_bind    8710..8731
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8746..8751,8752..8769,8770..8776)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8784..8800
                     /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     8789..8811
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8808..8824
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8808..8824
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(8825..8833,1..219)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MITNSSSVPGDPLESTCRHASLALAVVLQRRDWENPGVTQLNRLA
                     AHPPFASWRNSEEARTDRPSQQLRSLNGEC"
     misc_feature    join(8833,1..56)
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
ORIGIN
        1 aattcgagct cggtacccgg ggatcctcta gagtcgacct gcaggcatgc aagcttggca
       61 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
      121 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
      181 ccttcccaac agttgcgcag cctgaatggc gaatgctaga gcagcttgag cttggatcag
      241 attgtcgttt cccgccttca gtttaaacta tcagtgtttg acaggatata ttggcgggta
      301 aacctaagag aaaagagcgt ttattagaat aatcggatat ttaaaagggc gtgaaaaggt
      361 ttatccgttc gtccatttgt atgtgcatgc caaccacagg gttcccctcg ggatcaaagt
      421 actttgatcc aacccctccg ctgctatagt gcagtcggct tctgacgttc agtgcagccg
      481 tcttctgaaa acgacatgtc gcacaagtcc taagttacgc gacaggctgc cgccctgccc
      541 ttttcctggc gttttcttgt cgcgtgtttt agtcgcataa agtagaatac ttgcgactag
      601 aaccggagac attacgccat gaacaagagc gccgccgctg gcctgctggg ctatgcccgc
      661 gtcagcaccg acgaccagga cttgaccaac caacgggccg aactgcacgc ggccggctgc
      721 accaagctgt tttccgagaa gatcaccggc accaggcgcg accgcccgga gctggccagg
      781 atgcttgacc acctacgccc tggcgacgtt gtgacagtga ccaggctaga ccgcctggcc
      841 cgcagcaccc gcgacctact ggacattgcc gagcgcatcc aggaggccgg cgcgggcctg
      901 cgtagcctgg cagagccgtg ggccgacacc accacgccgg ccggccgcat ggtgttgacc
      961 gtgttcgccg gcattgccga gttcgagcgt tccctaatca tcgaccgcac ccggagcggg
     1021 cgcgaggccg ccaaggcccg aggcgtgaag tttggccccc gccctaccct caccccggca
     1081 cagatcgcgc acgcccgcga gctgatcgac caggaaggcc gcaccgtgaa agaggcggct
     1141 gcactgcttg gcgtgcatcg ctcgaccctg taccgcgcac ttgagcgcag cgaggaagtg
     1201 acgcccaccg aggccaggcg gcgcggtgcc ttccgtgagg acgcattgac cgaggccgac
     1261 gccctggcgg ccgccgagaa tgaacgccaa gaggaacaag catgaaaccg caccaggacg
     1321 gccaggacga accgtttttc attaccgaag agatcgaggc ggagatgatc gcggccgggt
     1381 acgtgttcga gccgcccgcg cacgtctcaa ccgtgcggct gcatgaaatc ctggccggtt
     1441 tgtctgatgc caagctggcg gcctggccgg ccagcttggc cgctgaagaa accgagcgcc
     1501 gccgtctaaa aaggtgatgt gtatttgagt aaaacagctt gcgtcatgcg gtcgctgcgt
     1561 atatgatgcg atgagtaaat aaacaaatac gcaaggggaa cgcatgaagg ttatcgctgt
     1621 acttaaccag aaaggcgggt caggcaagac gaccatcgca acccatctag cccgcgccct
     1681 gcaactcgcc ggggccgatg ttctgttagt cgattccgat ccccagggca gtgcccgcga
     1741 ttgggcggcc gtgcgggaag atcaaccgct aaccgttgtc ggcatcgacc gcccgacgat
     1801 tgaccgcgac gtgaaggcca tcggccggcg cgacttcgta gtgatcgacg gagcgcccca
     1861 ggcggcggac ttggctgtgt ccgcgatcaa ggcagccgac ttcgtgctga ttccggtgca
     1921 gccaagccct tacgacatat gggccaccgc cgacctggtg gagctggtta agcagcgcat
     1981 tgaggtcacg gatggaaggc tacaagcggc ctttgtcgtg tcgcgggcga tcaaaggcac
     2041 gcgcatcggc ggtgaggttg ccgaggcgct ggccgggtac gagctgccca ttcttgagtc
     2101 ccgtatcacg cagcgcgtga gctacccagg cactgccgcc gccggcacaa ccgttcttga
     2161 atcagaaccc gagggcgacg ctgcccgcga ggtccaggcg ctggccgctg aaattaaatc
     2221 aaaactcatt tgagttaatg aggtaaagag aaaatgagca aaagcacaaa cacgctaagt
     2281 gccggccgtc cgagcgcacg cagcagcaag gctgcaacgt tggccagcct ggcagacacg
     2341 ccagccatga agcgggtcaa ctttcagttg ccggcggagg atcacaccaa gctgaagatg
     2401 tacgcggtac gccaaggcaa gaccattacc gagctgctat ctgaatacat cgcgcagcta
     2461 ccagagtaaa tgagcaaatg aataaatgag tagatgaatt ttagcggcta aaggaggcgg
     2521 catggaaaat caagaacaac caggcaccga cgccgtggaa tgccccatgt gtggaggaac
     2581 gggcggttgg ccaggcgtaa gcggctgggt tgcctgccgg ccctgcaatg gcactggaac
     2641 ccccaagccc gaggaatcgg cgtgagcggt cgcaaaccat ccggcccggt acaaatcggc
     2701 gcggcgctgg gtgatgacct ggtggagaag ttgaaggccg cgcaggccgc ccagcggcaa
     2761 cgcatcgagg cagaagcacg ccccggtgaa tcgtggcaag cggccgctga tcgaatccgc
     2821 aaagaatccc ggcaaccgcc ggcagccggt gcgccgtcga ttaggaagcc gcccaagggc
     2881 gacgagcaac cagatttttt cgttccgatg ctctatgacg tgggcacccg cgatagtcgc
     2941 agcatcatgg acgtggccgt tttccgtctg tcgaagcgtg accgacgagc tggcgaggtg
     3001 atccgctacg agcttccaga cgggcacgta gaggtttccg cagggccggc cggcatggcc
     3061 agtgtgtggg attacgacct ggtactgatg gcggtttccc atctaaccga atccatgaac
     3121 cgataccggg aagggaaggg agacaagccc ggccgcgtgt tccgtccaca cgttgcggac
     3181 gtactcaagt tctgccggcg agccgatggc ggaaagcaga aagacgacct ggtagaaacc
     3241 tgcattcggt taaacaccac gcacgttgcc atgcagcgta cgaagaaggc caagaacggc
     3301 cgcctggtga cggtatccga gggtgaagcc ttgattagcc gctacaagat cgtaaagagc
     3361 gaaaccgggc ggccggagta catcgagatc gagctagctg attggatgta ccgcgagatc
     3421 acagaaggca agaacccgga cgtgctgacg gttcaccccg attacttttt gatcgatccc
     3481 ggcatcggcc gttttctcta ccgcctggca cgccgcgccg caggcaaggc agaagccaga
     3541 tggttgttca agacgatcta cgaacgcagt ggcagcgccg gagagttcaa gaagttctgt
     3601 ttcaccgtgc gcaagctgat cgggtcaaat gacctgccgg agtacgattt gaaggaggag
     3661 gcggggcagg ctggcccgat cctagtcatg cgctaccgca acctgatcga gggcgaagca
     3721 tccgccggtt cctaatgtac ggagcagatg ctagggcaaa ttgccctagc aggggaaaaa
     3781 ggtcgaaaag gtctctttcc tgtggatagc acgtacattg ggaacccaaa gccgtacatt
     3841 gggaaccgga acccgtacat tgggaaccca aagccgtaca ttgggaaccg gtcacacatg
     3901 taagtgactg atataaaaga gaaaaaaggc gatttttccg cctaaaactc tttaaaactt
     3961 attaaaactc ttaaaacccg cctggcctgt gcataactgt ctggccagcg cacagccgaa
     4021 gagctgcaaa aagcgcctac ccttcggtcg ctgcgctccc tacgccccgc cgcttcgcgt
     4081 cggcctatcg cggccgctgg ccgctcaaaa atggctggcc tacggccagg caatctacca
     4141 gggcgcggac aagccgcgcc gtcgccactc gaccgccggc gcccacatca aggcaccctg
     4201 cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     4261 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     4321 tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac
     4381 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa
     4441 ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc
     4501 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     4561 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     4621 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     4681 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     4741 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     4801 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     4861 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     4921 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     4981 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     5041 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     5101 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     5161 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     5221 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     5281 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgca ttctaggtac
     5341 taaaacaatt catccagtaa aatataatat tttattttct cccaatcagg cttgatcccc
     5401 agtaagtcaa aaaatagctc gacatactgt tcttccccga tatcctccct gatcgaccgg
     5461 acgcagaagg caatgtcata ccacttgtcc gccctgccgc ttctcccaag atcaataaag
     5521 ccacttactt tgccatcttt cacaaagatg ttgctgtctc ccaggtcgcc gtgggaaaag
     5581 acaagttcct cttcgggctt ttccgtcttt aaaaaatcat acagctcgcg cggatcttta
     5641 aatggagtgt cttcttccca gttttcgcaa tccacatcgg ccagatcgtt attcagtaag
     5701 taatccaatt cggctaagcg gctgtctaag ctattcgtat agggacaatc cgatatgtcg
     5761 atggagtgaa agagcctgat gcactccgca tacagctcga taatcttttc agggctttgt
     5821 tcatcttcat actcttccga gcaaaggacg ccatcggcct cactcatgag cagattgctc
     5881 cagccatcat gccgttcaaa gtgcaggacc tttggaacag gcagctttcc ttccagccat
     5941 agcatcatgt ccttttcccg ttccacatca taggtggtcc ctttataccg gctgtccgtc
     6001 atttttaaat ataggttttc attttctccc accagcttat ataccttagc aggagacatt
     6061 ccttccgtat cttttacgca gcggtatttt tcgatcagtt ttttcaattc cggtgatatt
     6121 ctcattttag ccatttatta tttccttcct cttttctaca gtatttaaag ataccccaag
     6181 aagctaatta taacaagacg aactccaatt cactgttcct tgcattctaa aaccttaaat
     6241 accagaaaac agctttttca aagttgtttt caaagttggc gtataacata gtatcgacgg
     6301 agccgatttt gaaaccgcgg tgatcacagg cagcaacgct ctgtcatcgt tacaatcaac
     6361 atgctaccct ccgcgagatc atccgtgttt caaacccggc agcttagttg ccgttcttcc
     6421 gaatagcatc ggtaacatga gcaaagtctg ccgccttaca acggctctcc cgctgacgcc
     6481 gtcccggact gatgggctgc ctgtatcgag tggtgatttt gtgccgagct gccggtcggg
     6541 gagctgttgg ctggctggtg gcaggatata ttgtggtgta aacaaattga cgcttagaca
     6601 acttaataac acattgcgga cgtttttaat gtactgaatt aacgccgaat taattcgggg
     6661 gatctggatt ttagtactgg attttggttt taggaattag aaattttatt gatagaagta
     6721 ttttacaaat acaaatacat actaagggtt tcttatatgc tcaacacatg agcgaaaccc
     6781 tataggaacc ctaattccct tatctgggaa ctactcacac attattatgg agaaactcga
     6841 gcttgtcgat cgactctagc tagaggatcg atccgaaccc cagagtcccg ctcagaagaa
     6901 ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga taccgtaaag
     6961 cacgaggaag cggtcagccc attcgccgcc aagctcttca gcaatatcac gggtagccaa
     7021 cgctatgtcc tgatagcggt ccgccacacc cagccggcca cagtcgatga atccagaaaa
     7081 gcggccattt tccaccatga tattcggcaa gcaggcatcg ccatgtgtca cgacgagatc
     7141 ctcgccgtcg ggcatgcgcg ccttgagcct ggcgaacagt tcggctggcg cgagcccctg
     7201 atgctcttcg tccagatcat cctgatcgac aagaccggct tccatccgag tacgtgctcg
     7261 ctcgatgcga tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa gcgtatgcag
     7321 ccgccgcatt gcatcagcca tgatggatac tttctcggca ggagcaaggt gagatgacag
     7381 gagatcctgc cccggcactt cgcccaatag cagccagtcc cttcccgctt cagtgacaac
     7441 gtcgagcaca gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc gcgctgcctc
     7501 gtcccctagg atcgatccat ggtcacaata gcagccagtc ccttcccgct tcagtgacaa
     7561 cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct
     7621 cgtcctggag ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc
     7681 cctgcgctga cagccgaaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt
     7741 catagccgaa tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt
     7801 caatccccat agagatagat ttgtagagag agactggtga tttcagcgtg tcctctccaa
     7861 atgaaatgaa cttccttata tagaggaagg gtcttgcgaa ggatagtggg attgtgcgtc
     7921 atcccttacg tcagtggaga tatcacatca atccacttgc tttgaagacg tggttggaac
     7981 gtcttctttt tccacgatgc tcctcgtggg tgggggtcca tctttgggac cactgtcggc
     8041 agaggcatct tgaacgatag cctttccttt atcgcaatga tggcatttgt aggtgccacc
     8101 ttccttttct actgtccttt tgatgaagtg acagatagct gggcaatgga atccgaggag
     8161 gtttcccgat attacccttt gttgaaaagt ctcaatagcc ctttggtctt ctgagactgt
     8221 atctttgata ttcttggagt agacgagagt gtcgtgctcc accatgttca catcaatcca
     8281 cttgctttga agacgtggtt ggaacgtctt ctttttccac gatgctcctc gtgggtgggg
     8341 gtccatcttt gggaccactg tcggcagagg catcttgaac gatagccttt cctttatcgc
     8401 aatgatggca tttgtaggtg ccaccttcct tttctactgt ccttttgatg aagtgacaga
     8461 tagctgggca atggaatccg aggaggtttc ccgatattac cctttgttga aaagtctcaa
     8521 tagccctttg gtcttctgag actgtatctt tgatattctt ggagtagacg agagtgtcgt
     8581 gctccaccat gttggcaagc tgctctagcc aatacgcaaa ccgcctctcc ccgcgcgttg
     8641 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     8701 caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct
     8761 tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta
     8821 tgacatgatt acg
//
