LOCUS       sequence-308825-        5763 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 1 position 1 containing the Nos promoter, NPTII (Kan 
            resistance), and the Nos terminator.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5763)
  AUTHORS   Grutzner R, Martin P, Horn C, Mortensen S, Cram EJ, Lee-Parsons CWT,
            Stuttmann J, Marillonnet S
  TITLE     High-efficiency genome editing in plants mediated by a Cas9 gene 
            containing multiple introns.
  JOURNAL   Plant Commun. 2020 Nov 23;2(2):100135. doi: 
            10.1016/j.xplc.2020.100135. eCollection 2021 Mar 8.
  PUBMED    33898975
REFERENCE   2  (bases 1 to 5763)
  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: "Plant 
            Commun."; date: "2020-11-23"; volume: "2(2):100135. doi"; pages: " 
            10.1016/j.xplc.2020.100135. eCollection 2021 Mar 8"
COMMENT     Sequence Label: pICH67131
FEATURES             Location/Qualifiers
     source          1..5763
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    46..70
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(226..1374)
                     /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(1710..2298)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1790..1809)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2469..3260,3261..3329))
                     /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     3092..3111
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3330..3434)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3521..4152
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    4244..4268
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     promoter        4453..4632
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     promoter        4485..4668
                     /function="NOS promoter"
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     CDS             4689..5483
                     /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(4743..4762)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5353..5372
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      5509..5761
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
ORIGIN
        1 cgctgcaatt gtcttctgca cgaagtggtt taaactatca gtgtttgaca ggatatattg
       61 gcgggtaaac ctaagagaaa agagcgttta ttagaataat cggatattta aaagggcgtg
      121 aaaaggttta tccgttcgtc catttgtatg tgcatgccaa ccacagggtt ccccagatca
      181 ggcgctggct gctgaacccc cagccggaac tgaccccaca aggccctagc gtttgcaatg
      241 caccaggtca tcattgaccc aggcgtgttc caccaggccg ctgcctcgca actcttcgca
      301 ggcttcgccg acctgctcgc gccacttctt cacgcgggtg gaatccgatc cgcacatgag
      361 gcggaaggtt tccagcttga gcgggtacgg ctcccggtgc gagctgaaat agtcgaacat
      421 ccgtcgggcc gtcggcgaca gcttgcggta cttctcccat atgaatttcg tgtagtggtc
      481 gccagcaaac agcacgacga tttcctcgtc gatcaggacc tggcaacggg acgttttctt
      541 gccacggtcc aggacgcgga agcggtgcag cagcgacacc gattccaggt gcccaacgcg
      601 gtcggacgtg aagcccatcg ccgtcgcctg taggcgcgac aggcattcct cggccttcgt
      661 gtaataccgg ccattgatcg accagcccag gtcctggcaa agctcgtaga acgtgaaggt
      721 gatcggctcg ccgatagggg tgcgcttcgc gtactccaac acctgctgcc acaccagttc
      781 gtcatcgtcg gcccgcagct cgacgccggt gtaggtgatc ttcacgtcct tgttgacgtg
      841 gaaaatgacc ttgttttgca gcgcctcgcg cgggattttc ttgttgcgcg tggtgaacag
      901 ggcagagcgg gccgtgtcgt ttggcatcgc tcgcatcgtg tccggccacg gcgcaatatc
      961 gaacaaggaa agctgcattt ccttgatctg ctgcttcgtg tgtttcagca acgcggcctg
     1021 cttggcctcg ctgacctgtt ttgccaggtc ctcgccggcg gtttttcgct tcttggtcgt
     1081 catagttcct cgcgtgtcga tggtcatcga cttcgccaaa cctgccgcct cctgttcaag
     1141 acgacgcgaa cgctccacgg cggccgatgg cgcgggcagg gcagggggag ccagttgcac
     1201 gctgtcgcgc tcgatcttgg ccgtagcttg ctggaccatc gagccgacgg actggaaggt
     1261 ttcgcggggc gcacgcatga cggtgcggct tgcgatggtt tcggcatcct cggcggaaaa
     1321 ccccgcgtcg atcagttctt gcctgtatgc cttccggtca aacgtccgat tcattcaccc
     1381 tccttgcggg attgccccga ctcacgccgg ggcaatgtgc ccttattcct gatttgaccc
     1441 gcctggtgcc ttggtgtcca gataatccac cttatcggca atgaagtcgg tcccgtagac
     1501 cgtctggccg tccttctcgt acttggtatt ccgaatcttg ccctgcacga ataccagcga
     1561 ccccttgccc aaatacttgc cgtgggcctc ggcctgagag ccaaaacact tgatgcggaa
     1621 gaagtcggtg cgctcctgct tgtcgccggc atcgttgcgc cacatctagg atctgccagg
     1681 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     1741 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     1801 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     1861 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     1921 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     1981 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     2041 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     2101 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     2161 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     2221 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     2281 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     2341 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     2401 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     2461 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     2521 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     2581 tctggcccca gtgctgcaat gataccgcga gaaccacgct caccggctcc agatttatca
     2641 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     2701 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     2761 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     2821 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     2881 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     2941 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     3001 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     3061 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     3121 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     3181 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     3241 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     3301 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     3361 tatcaaggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     3421 ataggggttc cgcgcacgaa ttggccagcg ctgccatttt tggggtgagg ccgttcgcgg
     3481 ccgaggggcg cagcccctgg ggggatggga ggcccgcgtt agcgggccgg gagggttcga
     3541 gaaggggggg cacccccctt cggcgtgcgc ggtcacgcgc acagggcgca gccctggtta
     3601 aaaacaaggt ttataaatat tggtttaaaa gcaggttaaa agacaggtta gcggtggccg
     3661 aaaaacgggc ggaaaccctt gcaaatgctg gattttctgc ctgtggacag cccctcaaat
     3721 gtcaataggt gcgcccctca tctgtcagca ctctgcccct caagtgtcaa ggatcgcgcc
     3781 cctcatctgt cagtagtcgc gcccctcaag tgtcaatacc gcagggcact tatccccagg
     3841 cttgtccaca tcatctgtgg gaaactcgcg taaaatcagg cgttttcgcc gatttgcgag
     3901 gctggccagc tccacgtcgc cggccgaaat cgagcctgcc cctcatctgt caacgccgcg
     3961 ccgggtgagt cggcccctca agtgtcaacg tccgcccctc atctgtcagt gagggccaag
     4021 ttttccgcga ggtatccaca acgccggcgg ccgcggtgtc tcgcacacgg cttcgacggc
     4081 gtttctggcg cgtttgcagg gccatagacg gccgccagcc cagcggcgag ggcaaccagc
     4141 ccggtgagcg tcgcaaagga gatcctgatc tgactgatgg gctgcctgta tcgagtggtg
     4201 attttgtgcc gagctgccgg tcggggagct gttggctggc tggtggcagg atatattgtg
     4261 gtgtaaacaa attgacgctt agacaactta ataacacatt gcggacgttt ttaatgtact
     4321 ggggtggatg cagtgggccc cactctgtga agacaatgcc gaattcggat ccggagagcg
     4381 gagaattaag ggagtcacgt tatgaccccc gccgatgacg cgggacaagc cgttttacgt
     4441 ttggaactga cagaaccgca acgttgaagg agccactcag ccgcgggttt ctggagttta
     4501 atgagctaag cacatacgtc agaaaccatt attgcgcgtt caaaagtcgc ctaaggtcac
     4561 tatcagctag caaatatttc ttgtcaaaaa tgctccactg acgttccata aattcccctc
     4621 ggtatccaat tagagtctca tattcactct caatccaaat aatctgcacc ggatctggat
     4681 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     4741 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     4801 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     4861 atgaactcca ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     4921 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     4981 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     5041 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     5101 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     5161 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgta
     5221 tgcccgacgg cgaggatctc gtcgtgactc atggcgatgc ctgcttgccg aatatcatgg
     5281 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     5341 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     5401 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     5461 gccttcttga cgagttcttc tgagcgggtc tggatctaga gtcaagcaga tcgttcaaac
     5521 atttggcaat aaagtttctt aagattgaat cctgttgccg gtcttgcgat gattatcata
     5581 taatttctgt tgaattacgt taagcatgta ataattaaca tgtaatgcat gacgttattt
     5641 atgagatggg tttttatgat tagagtcccg caattataca tttaatacgc gatagaaaac
     5701 aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg tgtcatctat gttactagat
     5761 cga
//
