LOCUS       sequence-119197-        6278 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 1 Golden Gate Cassette: Plant kanamycin resistance cassette .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6278)
  AUTHORS   Lawrenson T, Shorinola O, Stacey N, Li C, Ostergaard L, Patron N, 
            Uauy C, Harwood W
  TITLE     Induction of targeted, heritable mutations in barley and Brassica 
            oleracea using RNA-guided Cas9 nuclease.
  JOURNAL   Genome Biol. 2015 Nov 30;16(1):258. doi: 10.1186/s13059-015-0826-7.
  PUBMED    26616834
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: "Genome 
            Biol."; date: "2015-11-30"; volume: "16(1):258. doi"; pages: " 
            10.1186/s13059-015-0826-7"
COMMENT     Sequence Label: pICSL11055
FEATURES             Location/Qualifiers
     source          1..6278
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        438..783
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     726..746
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     CDS             874..1668
                     /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(928..947)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1538..1557
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      1681..1933
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     misc_feature    1981..2005
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(2161..3309)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     rep_origin      complement(3645..4233)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3725..3744)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4404..5195,5196..5264))
                     /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     5027..5046
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5265..5369)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5456..6087
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    6179..6203
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 tctgtgaaga caatgccgaa ttcggatccg gaggtcaaca tggtggagca cgacactctg
       61 gtctactcca aaaatgtcaa agatacagtc tcagaagatc aaagggctat tgagactttt
      121 caacaaagga taatttcggg aaacctcctc ggattccatt gcccagctat ctgtcacttc
      181 atcgaaagga cagtagaaaa ggaaggtggc tcctacaaat gccatcattg cgataaagga
      241 aaggctatca ttcaagatct ctctgccgac agtggtccca aagatggacc cccacccacg
      301 aggagcatcg tggaaaaaga agaggttcca accacgtcta caaagcaagt ggattgatgt
      361 gataacatgg tggagcacga cactctggtc tactccaaaa atgtcaaaga tacagtctca
      421 gaagatcaaa gggctattga gacttttcaa caaaggataa tttcgggaaa cctcctcgga
      481 ttccattgcc cagctatctg tcacttcatc gaaaggacag tagaaaagga aggtggctcc
      541 tacaaatgcc atcattgcga taaaggaaag gctatcattc aagatctctc tgccgacagt
      601 ggtcccaaag atggaccccc acccacgagg agcatcgtgg aaaaagaaga ggttccaacc
      661 acgtctacaa agcaagtgga ttgatgtgac atctccactg acgtaaggga tgacgcacaa
      721 tcccactatc cttcgcaaga cccttcctct atataaggaa gttcatttca tttggagagg
      781 acacgctcga gtataagagc tcatttttac aacaattacc aacaacaaca aacaacaaac
      841 aacattacaa ttacatttac aattatcgat acaatgattg aacaagatgg attgcacgca
      901 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
      961 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     1021 aagaccgacc tgtccggtgc cctgaatgaa ctccaagacg aggcagcgcg gctatcgtgg
     1081 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     1141 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     1201 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     1261 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     1321 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     1381 ctgttcgcca ggctcaaggc gcggatgccc gacggcgagg atctcgtcgt gacccacggc
     1441 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     1501 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     1561 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     1621 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc ttgtcaagca
     1681 gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg
     1741 atgattatca tataatttct gttgaattac gttaagcatg taataattaa catgtaatgc
     1801 atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata catttaatac
     1861 gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct
     1921 atgttactag atcgacgctg caattgtctt ctgcacgaag tggtttaaac tatcagtgtt
     1981 tgacaggata tattggcggg taaacctaag agaaaagagc gtttattaga ataatcggat
     2041 atttaaaagg gcgtgaaaag gtttatccgt tcgtccattt gtatgtgcat gccaaccaca
     2101 gggttcccca gatcaggcgc tggctgctga acccccagcc ggaactgacc ccacaaggcc
     2161 ctagcgtttg caatgcacca ggtcatcatt gacccaggcg tgttccacca ggccgctgcc
     2221 tcgcaactct tcgcaggctt cgccgacctg ctcgcgccac ttcttcacgc gggtggaatc
     2281 cgatccgcac atgaggcgga aggtttccag cttgagcggg tacggctccc ggtgcgagct
     2341 gaaatagtcg aacatccgtc gggccgtcgg cgacagcttg cggtacttct cccatatgaa
     2401 tttcgtgtag tggtcgccag caaacagcac gacgatttcc tcgtcgatca ggacctggca
     2461 acgggacgtt ttcttgccac ggtccaggac gcggaagcgg tgcagcagcg acaccgattc
     2521 caggtgccca acgcggtcgg acgtgaagcc catcgccgtc gcctgtaggc gcgacaggca
     2581 ttcctcggcc ttcgtgtaat accggccatt gatcgaccag cccaggtcct ggcaaagctc
     2641 gtagaacgtg aaggtgatcg gctcgccgat aggggtgcgc ttcgcgtact ccaacacctg
     2701 ctgccacacc agttcgtcat cgtcggcccg cagctcgacg ccggtgtagg tgatcttcac
     2761 gtccttgttg acgtggaaaa tgaccttgtt ttgcagcgcc tcgcgcggga ttttcttgtt
     2821 gcgcgtggtg aacagggcag agcgggccgt gtcgtttggc atcgctcgca tcgtgtccgg
     2881 ccacggcgca atatcgaaca aggaaagctg catttccttg atctgctgct tcgtgtgttt
     2941 cagcaacgcg gcctgcttgg cctcgctgac ctgttttgcc aggtcctcgc cggcggtttt
     3001 tcgcttcttg gtcgtcatag ttcctcgcgt gtcgatggtc atcgacttcg ccaaacctgc
     3061 cgcctcctgt tcaagacgac gcgaacgctc cacggcggcc gatggcgcgg gcagggcagg
     3121 gggagccagt tgcacgctgt cgcgctcgat cttggccgta gcttgctgga ccatcgagcc
     3181 gacggactgg aaggtttcgc ggggcgcacg catgacggtg cggcttgcga tggtttcggc
     3241 atcctcggcg gaaaaccccg cgtcgatcag ttcttgcctg tatgccttcc ggtcaaacgt
     3301 ccgattcatt caccctcctt gcgggattgc cccgactcac gccggggcaa tgtgccctta
     3361 ttcctgattt gacccgcctg gtgccttggt gtccagataa tccaccttat cggcaatgaa
     3421 gtcggtcccg tagaccgtct ggccgtcctt ctcgtacttg gtattccgaa tcttgccctg
     3481 cacgaatacc agcgacccct tgcccaaata cttgccgtgg gcctcggcct gagagccaaa
     3541 acacttgatg cggaagaagt cggtgcgctc ctgcttgtcg ccggcatcgt tgcgccacat
     3601 ctaggatctg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     3661 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     3721 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     3781 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     3841 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     3901 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     3961 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     4021 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     4081 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     4141 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     4201 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     4261 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     4321 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     4381 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     4441 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     4501 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaacc acgctcaccg
     4561 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     4621 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     4681 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     4741 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     4801 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     4861 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     4921 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     4981 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     5041 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     5101 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     5161 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     5221 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     5281 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     5341 tagaaaaata aacaaatagg ggttccgcgc acgaattggc cagcgctgcc atttttgggg
     5401 tgaggccgtt cgcggccgag gggcgcagcc cctgggggga tgggaggccc gcgttagcgg
     5461 gccgggaggg ttcgagaagg gggggcaccc cccttcggcg tgcgcggtca cgcgcacagg
     5521 gcgcagccct ggttaaaaac aaggtttata aatattggtt taaaagcagg ttaaaagaca
     5581 ggttagcggt ggccgaaaaa cgggcggaaa cccttgcaaa tgctggattt tctgcctgtg
     5641 gacagcccct caaatgtcaa taggtgcgcc cctcatctgt cagcactctg cccctcaagt
     5701 gtcaaggatc gcgcccctca tctgtcagta gtcgcgcccc tcaagtgtca ataccgcagg
     5761 gcacttatcc ccaggcttgt ccacatcatc tgtgggaaac tcgcgtaaaa tcaggcgttt
     5821 tcgccgattt gcgaggctgg ccagctccac gtcgccggcc gaaatcgagc ctgcccctca
     5881 tctgtcaacg ccgcgccggg tgagtcggcc cctcaagtgt caacgtccgc ccctcatctg
     5941 tcagtgaggg ccaagttttc cgcgaggtat ccacaacgcc ggcggccgcg gtgtctcgca
     6001 cacggcttcg acggcgtttc tggcgcgttt gcagggccat agacggccgc cagcccagcg
     6061 gcgagggcaa ccagcccggt gagcgtcgca aaggagatcc tgatctgact gatgggctgc
     6121 ctgtatcgag tggtgatttt gtgccgagct gccggtcggg gagctgttgg ctggctggtg
     6181 gcaggatata ttgtggtgta aacaaattga cgcttagaca acttaataac acattgcgga
     6241 cgtttttaat gtactggggt ggatgcagtg ggccccac
//
