LOCUS       sequence-377099-        6278 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Kanamycin resistance in plants.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6278)
  AUTHORS   Lawrenson T, Youles M, Chhetry M, Clarke M, Harwood W, Hundleby P
  TITLE     Efficient Targeted Mutagenesis in Brassica Crops Using CRISPR/Cas 
            Systems.
  JOURNAL   Methods Mol Biol. 2023;2653:253-271. doi: 
            10.1007/978-1-0716-3131-7_16.
  PUBMED    36995631
REFERENCE   2  (bases 1 to 6278)
  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: "Methods Mol
            Biol."; date: "2023"; volume: "2653:253-271. doi"; pages: " 
            10.1007/978-1-0716-3131-7_16"
COMMENT     Sequence Label: pICSL11137
FEATURES             Location/Qualifiers
     source          1..6278
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    74..98
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     rep_origin      190..821
                     /label=oriV
                     /note="incP origin of replication"
     promoter        908..1012
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1013..1081,1082..1873)
                     /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(1231..1250)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2044..2632
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2533..2552
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             2968..4116
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     misc_feature    4272..4296
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     promoter        4746..5091
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     5034..5054
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     CDS             5182..5976
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5236..5255)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5846..5865
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      5989..6241
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
ORIGIN
        1 ggggcccact gcatccaccc cagtacatta aaaacgtccg caatgtgtta ttaagttgtc
       61 taagcgtcaa tttgtttaca ccacaatata tcctgccacc agccagccaa cagctccccg
      121 accggcagct cggcacaaaa tcaccactcg atacaggcag cccatcagtc agatcaggat
      181 ctcctttgcg acgctcaccg ggctggttgc cctcgccgct gggctggcgg ccgtctatgg
      241 ccctgcaaac gcgccagaaa cgccgtcgaa gccgtgtgcg agacaccgcg gccgccggcg
      301 ttgtggatac ctcgcggaaa acttggccct cactgacaga tgaggggcgg acgttgacac
      361 ttgaggggcc gactcacccg gcgcggcgtt gacagatgag gggcaggctc gatttcggcc
      421 ggcgacgtgg agctggccag cctcgcaaat cggcgaaaac gcctgatttt acgcgagttt
      481 cccacagatg atgtggacaa gcctggggat aagtgccctg cggtattgac acttgagggg
      541 cgcgactact gacagatgag gggcgcgatc cttgacactt gaggggcaga gtgctgacag
      601 atgaggggcg cacctattga catttgaggg gctgtccaca ggcagaaaat ccagcatttg
      661 caagggtttc cgcccgtttt tcggccaccg ctaacctgtc ttttaacctg cttttaaacc
      721 aatatttata aaccttgttt ttaaccaggg ctgcgccctg tgcgcgtgac cgcgcacgcc
      781 gaaggggggt gccccccctt ctcgaaccct cccggcccgc taacgcgggc ctcccatccc
      841 cccaggggct gcgcccctcg gccgcgaacg gcctcacccc aaaaatggca gcgctggcca
      901 attcgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
      961 catgagacaa taaccttgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     1021 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     1081 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     1141 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     1201 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     1261 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     1321 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     1381 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     1441 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     1501 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     1561 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     1621 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     1681 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggtt ctcgcggtat
     1741 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     1801 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     1861 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     1921 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     1981 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     2041 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     2101 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     2161 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     2221 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     2281 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     2341 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     2401 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     2461 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     2521 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     2581 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     2641 caacgcggcc tttttacggt tcctggcaga tcctagatgt ggcgcaacga tgccggcgac
     2701 aagcaggagc gcaccgactt cttccgcatc aagtgttttg gctctcaggc cgaggcccac
     2761 ggcaagtatt tgggcaaggg gtcgctggta ttcgtgcagg gcaagattcg gaataccaag
     2821 tacgagaagg acggccagac ggtctacggg accgacttca ttgccgataa ggtggattat
     2881 ctggacacca aggcaccagg cgggtcaaat caggaataag ggcacattgc cccggcgtga
     2941 gtcggggcaa tcccgcaagg agggtgaatg aatcggacgt ttgaccggaa ggcatacagg
     3001 caagaactga tcgacgcggg gttttccgcc gaggatgccg aaaccatcgc aagccgcacc
     3061 gtcatgcgtg cgccccgcga aaccttccag tccgtcggct cgatggtcca gcaagctacg
     3121 gccaagatcg agcgcgacag cgtgcaactg gctccccctg ccctgcccgc gccatcggcc
     3181 gccgtggagc gttcgcgtcg tcttgaacag gaggcggcag gtttggcgaa gtcgatgacc
     3241 atcgacacgc gaggaactat gacgaccaag aagcgaaaaa ccgccggcga ggacctggca
     3301 aaacaggtca gcgaggccaa gcaggccgcg ttgctgaaac acacgaagca gcagatcaag
     3361 gaaatgcagc tttccttgtt cgatattgcg ccgtggccgg acacgatgcg agcgatgcca
     3421 aacgacacgg cccgctctgc cctgttcacc acgcgcaaca agaaaatccc gcgcgaggcg
     3481 ctgcaaaaca aggtcatttt ccacgtcaac aaggacgtga agatcaccta caccggcgtc
     3541 gagctgcggg ccgacgatga cgaactggtg tggcagcagg tgttggagta cgcgaagcgc
     3601 acccctatcg gcgagccgat caccttcacg ttctacgagc tttgccagga cctgggctgg
     3661 tcgatcaatg gccggtatta cacgaaggcc gaggaatgcc tgtcgcgcct acaggcgacg
     3721 gcgatgggct tcacgtccga ccgcgttggg cacctggaat cggtgtcgct gctgcaccgc
     3781 ttccgcgtcc tggaccgtgg caagaaaacg tcccgttgcc aggtcctgat cgacgaggaa
     3841 atcgtcgtgc tgtttgctgg cgaccactac acgaaattca tatgggagaa gtaccgcaag
     3901 ctgtcgccga cggcccgacg gatgttcgac tatttcagct cgcaccggga gccgtacccg
     3961 ctcaagctgg aaaccttccg cctcatgtgc ggatcggatt ccacccgcgt gaagaagtgg
     4021 cgcgagcagg tcggcgaagc ctgcgaagag ttgcgaggca gcggcctggt ggaacacgcc
     4081 tgggtcaatg atgacctggt gcattgcaaa cgctagggcc ttgtggggtc agttccggct
     4141 gggggttcag cagccagcgc ctgatctggg gaaccctgtg gttggcatgc acatacaaat
     4201 ggacgaacgg ataaaccttt tcacgccctt ttaaatatcc gattattcta ataaacgctc
     4261 ttttctctta ggtttacccg ccaatatatc ctgtcaaaca ctgatagttt aaaccacttc
     4321 gtgcagaaga caattgcgga ggtcaacatg gtggagcacg acactctggt ctactccaaa
     4381 aatgtcaaag atacagtctc agaagatcaa agggctattg agacttttca acaaaggata
     4441 atttcgggaa acctcctcgg attccattgc ccagctatct gtcacttcat cgaaaggaca
     4501 gtagaaaagg aaggtggctc ctacaaatgc catcattgcg ataaaggaaa ggctatcatt
     4561 caagatctct ctgccgacag tggtcccaaa gatggacccc cacccacgag gagcatcgtg
     4621 gaaaaagaag aggttccaac cacgtctaca aagcaagtgg attgatgtga taacatggtg
     4681 gagcacgaca ctctggtcta ctccaaaaat gtcaaagata cagtctcaga agatcaaagg
     4741 gctattgaga cttttcaaca aaggataatt tcgggaaacc tcctcggatt ccattgccca
     4801 gctatctgtc acttcatcga aaggacagta gaaaaggaag gtggctccta caaatgccat
     4861 cattgcgata aaggaaaggc tatcattcaa gatctctctg ccgacagtgg tcccaaagat
     4921 ggacccccac ccacgaggag catcgtggaa aaagaagagg ttccaaccac gtctacaaag
     4981 caagtggatt gatgtgacat ctccactgac gtaagggatg acgcacaatc ccactatcct
     5041 tcgcaagacc cttcctctat ataaggaagt tcatttcatt tggagaggac acgctcgagt
     5101 ataagagctc atttttacaa caattaccaa caacaacaaa caacaaacaa cattacaatt
     5161 acatttacaa ttatcgatac aatgattgaa caagatggat tgcacgcagg ttctccggcc
     5221 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     5281 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     5341 tccggtgccc tgaatgaact ccaagacgag gcagcgcggc tatcgtggct ggccacgacg
     5401 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     5461 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     5521 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     5581 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     5641 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     5701 ctcaaggcgc ggatgcccga cggcgaggat ctcgtcgtga cccacggcga tgcctgcttg
     5761 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     5821 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     5881 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     5941 atcgccttct atcgccttct tgacgagttc ttctgagctt gtcaagcaga tcgttcaaac
     6001 atttggcaat aaagtttctt aagattgaat cctgttgccg gtcttgcgat gattatcata
     6061 taatttctgt tgaattacgt taagcatgta ataattaaca tgtaatgcat gacgttattt
     6121 atgagatggg tttttatgat tagagtcccg caattataca tttaatacgc gatagaaaac
     6181 aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg tgtcatctat gttactagat
     6241 cgacgctgga tccgaattcg gcattgtctt cacagagt
//
