LOCUS       sequence-228090-        9551 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 1 Golden Gate Cassette: eSaCas9 expression cassette for 
            dicotyledonous plants.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9551)
  AUTHORS   Raitskin O, Schudoma C, West A, Patron NJ
  TITLE     Comparison of efficiency and specificity of CRISPR-associated (Cas) 
            nucleases in plants: An expanded toolkit for precision genome 
            engineering.
  JOURNAL   PLoS One. 2019 Feb 27;14(2):e0211598. doi: 
            10.1371/journal.pone.0211598. eCollection 2019.
  PUBMED    30811422
REFERENCE   2  (bases 1 to 9551)
  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: "PLoS One.";
            date: "2019-02-27"; volume: "14(2):e0211598. doi"; pages: " 
            10.1371/journal.pone.0211598. eCollection 2019"
COMMENT     Sequence Label: pEPOR1CB0113
FEATURES             Location/Qualifiers
     source          1..9551
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    42..66
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(222..1370)
                     /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(1706..2294)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1786..1805)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2465..3256,3257..3325))
                     /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     3088..3107
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3326..3430)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3517..4148
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    4240..4264
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     promoter        4777..5122
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     5065..5085
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     CDS             5219..5239
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8420..8467
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             8474..8554
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             join(8561..8563,8564..8566,8567..9280)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(8606..8627)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(8867..8886)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     9214..9235
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     terminator      9293..9545
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
ORIGIN
        1 actattgtct tctgcacgaa gtggtttaaa ctatcagtgt ttgacaggat atattggcgg
       61 gtaaacctaa gagaaaagag cgtttattag aataatcgga tatttaaaag ggcgtgaaaa
      121 ggtttatccg ttcgtccatt tgtatgtgca tgccaaccac agggttcccc agatcaggcg
      181 ctggctgctg aacccccagc cggaactgac cccacaaggc cctagcgttt gcaatgcacc
      241 aggtcatcat tgacccaggc gtgttccacc aggccgctgc ctcgcaactc ttcgcaggct
      301 tcgccgacct gctcgcgcca cttcttcacg cgggtggaat ccgatccgca catgaggcgg
      361 aaggtttcca gcttgagcgg gtacggctcc cggtgcgagc tgaaatagtc gaacatccgt
      421 cgggccgtcg gcgacagctt gcggtacttc tcccatatga atttcgtgta gtggtcgcca
      481 gcaaacagca cgacgatttc ctcgtcgatc aggacctggc aacgggacgt tttcttgcca
      541 cggtccagga cgcggaagcg gtgcagcagc gacaccgatt ccaggtgccc aacgcggtcg
      601 gacgtgaagc ccatcgccgt cgcctgtagg cgcgacaggc attcctcggc cttcgtgtaa
      661 taccggccat tgatcgacca gcccaggtcc tggcaaagct cgtagaacgt gaaggtgatc
      721 ggctcgccga taggggtgcg cttcgcgtac tccaacacct gctgccacac cagttcgtca
      781 tcgtcggccc gcagctcgac gccggtgtag gtgatcttca cgtccttgtt gacgtggaaa
      841 atgaccttgt tttgcagcgc ctcgcgcggg attttcttgt tgcgcgtggt gaacagggca
      901 gagcgggccg tgtcgtttgg catcgctcgc atcgtgtccg gccacggcgc aatatcgaac
      961 aaggaaagct gcatttcctt gatctgctgc ttcgtgtgtt tcagcaacgc ggcctgcttg
     1021 gcctcgctga cctgttttgc caggtcctcg ccggcggttt ttcgcttctt ggtcgtcata
     1081 gttcctcgcg tgtcgatggt catcgacttc gccaaacctg ccgcctcctg ttcaagacga
     1141 cgcgaacgct ccacggcggc cgatggcgcg ggcagggcag ggggagccag ttgcacgctg
     1201 tcgcgctcga tcttggccgt agcttgctgg accatcgagc cgacggactg gaaggtttcg
     1261 cggggcgcac gcatgacggt gcggcttgcg atggtttcgg catcctcggc ggaaaacccc
     1321 gcgtcgatca gttcttgcct gtatgccttc cggtcaaacg tccgattcat tcaccctcct
     1381 tgcgggattg ccccgactca cgccggggca atgtgccctt attcctgatt tgacccgcct
     1441 ggtgccttgg tgtccagata atccacctta tcggcaatga agtcggtccc gtagaccgtc
     1501 tggccgtcct tctcgtactt ggtattccga atcttgccct gcacgaatac cagcgacccc
     1561 ttgcccaaat acttgccgtg ggcctcggcc tgagagccaa aacacttgat gcggaagaag
     1621 tcggtgcgct cctgcttgtc gccggcatcg ttgcgccaca tctaggatct gccaggaacc
     1681 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     1741 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     1801 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     1861 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     1921 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     1981 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     2041 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     2101 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     2161 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     2221 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     2281 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     2341 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     2401 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     2461 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     2521 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     2581 gccccagtgc tgcaatgata ccgcgagaac cacgctcacc ggctccagat ttatcagcaa
     2641 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     2701 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     2761 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     2821 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     2881 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     2941 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     3001 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     3061 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     3121 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     3181 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     3241 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     3301 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     3361 aaggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     3421 gggttccgcg cacgaattgg ccagcgctgc catttttggg gtgaggccgt tcgcggccga
     3481 ggggcgcagc ccctgggggg atgggaggcc cgcgttagcg ggccgggagg gttcgagaag
     3541 ggggggcacc ccccttcggc gtgcgcggtc acgcgcacag ggcgcagccc tggttaaaaa
     3601 caaggtttat aaatattggt ttaaaagcag gttaaaagac aggttagcgg tggccgaaaa
     3661 acgggcggaa acccttgcaa atgctggatt ttctgcctgt ggacagcccc tcaaatgtca
     3721 ataggtgcgc ccctcatctg tcagcactct gcccctcaag tgtcaaggat cgcgcccctc
     3781 atctgtcagt agtcgcgccc ctcaagtgtc aataccgcag ggcacttatc cccaggcttg
     3841 tccacatcat ctgtgggaaa ctcgcgtaaa atcaggcgtt ttcgccgatt tgcgaggctg
     3901 gccagctcca cgtcgccggc cgaaatcgag cctgcccctc atctgtcaac gccgcgccgg
     3961 gtgagtcggc ccctcaagtg tcaacgtccg cccctcatct gtcagtgagg gccaagtttt
     4021 ccgcgaggta tccacaacgc cggcggccgc ggtgtctcgc acacggcttc gacggcgttt
     4081 ctggcgcgtt tgcagggcca tagacggccg ccagcccagc ggcgagggca accagcccgg
     4141 tgagcgtcgc aaaggagatc ctgatctgac tgatgggctg cctgtatcga gtggtgattt
     4201 tgtgccgagc tgccggtcgg ggagctgttg gctggctggt ggcaggatat attgtggtgt
     4261 aaacaaattg acgcttagac aacttaataa cacattgcgg acgtttttaa tgtactgggg
     4321 tggatgcagt gggccccact ctgtgaagac aagcaagaat tcaagcttgg aggtcaacat
     4381 ggtggagcac gacactctgg tctactccaa aaatgtcaaa gatacagtct cagaagatca
     4441 aagggctatt gagacttttc aacaaaggat aatttcggga aacctcctcg gattccattg
     4501 cccagctatc tgtcacttca tcgaaaggac agtagaaaag gaaggtggct cctacaaatg
     4561 ccatcattgc gataaaggaa aggctatcat tcaagatctc tctgccgaca gtggtcccaa
     4621 agatggaccc ccacccacga ggagcatcgt ggaaaaagaa gaggttccaa ccacgtctac
     4681 aaagcaagtg gattgatgtg ataacatggt ggagcacgac actctggtct actccaaaaa
     4741 tgtcaaagat acagtctcag aagatcaaag ggctattgag acttttcaac aaaggataat
     4801 ttcgggaaac ctcctcggat tccattgccc agctatctgt cacttcatcg aaaggacagt
     4861 agaaaaggaa ggtggctcct acaaatgcca tcattgcgat aaaggaaagg ctatcattca
     4921 agatctctct gccgacagtg gtcccaaaga tggaccccca cccacgagga gcatcgtgga
     4981 aaaagaagag gttccaacca cgtctacaaa gcaagtggat tgatgtgaca tctccactga
     5041 cgtaagggat gacgcacaat cccactatcc ttcgcaagac ccttcctcta tataaggaag
     5101 ttcatttcat ttggagagga cacgctcgag tataagagct catttttaca acaattacca
     5161 acaacaacaa acaacaaaca acattacaat tacatttaca attatcgata caatggcccc
     5221 aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccaagcgga actacatcct
     5281 gggcctggac atcggcatca ccagcgtggg ctacggcatc atcgactacg agacacggga
     5341 cgtgatcgat gccggcgtgc ggctgttcaa agaggccaac gtggaaaaca acgagggcag
     5401 gcggagcaag agaggcgcca gaaggctgaa gcggcggagg cggcatagaa tccagagagt
     5461 gaagaagctg ctgttcgact acaacctgct gaccgaccac agcgagctga gcggcatcaa
     5521 cccctacgag gccagagtga agggcctgag ccagaagctg agcgaggaag agttctctgc
     5581 cgccctgctg cacctggcca agagaagagg cgtgcacaac gtgaacgagg tggaagagga
     5641 caccggcaac gagctgtcca ccaaagagca gatcagccgg aacagcaagg ccctggaaga
     5701 gaaatacgtg gccgaactgc agctggaacg gctgaagaaa gacggcgaag tgcggggcag
     5761 catcaacaga ttcaagacca gcgactacgt gaaagaagcc aaacagctgc tgaaggtgca
     5821 gaaggcctac caccagctgg accagagctt catcgacacc tacatcgacc tgctggaaac
     5881 ccggcggacc tactatgagg gacctggcga gggcagcccc ttcggctgga aggacatcaa
     5941 agaatggtac gagatgctga tgggccactg cacctacttc cccgaggaac tgcggagcgt
     6001 gaagtacgcc tacaacgccg acctgtacaa cgccctgaac gacctgaaca atctcgtgat
     6061 caccagggac gagaacgaga agctggaata ttacgagaag ttccagatca tcgagaacgt
     6121 gttcaagcag aagaagaagc ccaccctgaa gcagatcgcc aaagaaatcc tcgtgaacga
     6181 agaggatatt aagggctaca gagtgaccag caccggcaag cccgagttca ccaacctgaa
     6241 ggtgtaccac gacatcaagg acattaccgc ccggaaagag attattgaga acgccgagct
     6301 gctggatcag attgccaaga tcctgaccat ctaccagagc agcgaggaca tccaggaaga
     6361 actgaccaat ctgaactccg agctgaccca ggaagagatc gagcagatct ctaatctgaa
     6421 gggctatacc ggcacccaca acctgagcct gaaggccatc aacctgatcc tggacgagct
     6481 gtggcacacc aacgacaacc agatcgctat cttcaaccgg ctgaagctgg tgcccaagaa
     6541 ggtggacctg tcccagcaga aagagatccc caccaccctg gtggacgact tcatcctgag
     6601 ccccgtcgtg aagagaagct tcatccagag catcaaagtg atcaacgcca tcatcaagaa
     6661 gtacggcctg cccaacgaca tcattatcga gctggcccgc gagaagaact ccaaggacgc
     6721 ccagaaaatg atcaacgaga tgcagaagcg gaacgcgaaa accaacgagc ggatcgagga
     6781 aatcatccgg accaccggca aagagaacgc caagtacctg atcgagaaga tcaagctgca
     6841 cgacatgcag gaaggcaagt gcctgtacag cctggaagcc atccctctgg aagatctgct
     6901 gaacaacccc ttcaactatg aggtggacca catcatcccc agaagcgtgt ccttcgacaa
     6961 cagcttcaac aacaaggtgc tcgtgaagca ggaagaaaac agcaagaagg gcaaccggac
     7021 cccattccag tacctgagca gcagcgacag caagatcagc tacgaaacct tcaagaagca
     7081 catcctgaat ctggccaagg gcaagggcag aatcagcaag accaagaaag agtatctgct
     7141 ggaagaacgg gacatcaaca ggttctccgt gcagaaagac ttcatcaacc ggaacctggt
     7201 ggataccaga tacgccaccg cacgtctgat gaacctgctg cggagctact tcagagtgaa
     7261 caacctggac gtgaaagtga agtccatcaa tggcggcttc accagctttc tgcggcggaa
     7321 gtggaagttt aagaaagagc ggaacaaggg gtacaagcac cacgccgagg acgccctgat
     7381 cattgccaac gccgatttca tcttcaaaga gtggaagaaa ctggacaagg ccaaaaaagt
     7441 gatggaaaac cagatgttcg aggaaaagca ggccgagagc atgcccgaga tcgaaaccga
     7501 gcaggagtac aaagagatct tcatcacccc ccaccagatc aagcacatta aggacttcaa
     7561 ggactacaag tacagccacc gggtggacaa gaagcctaat agagagctga ttaacgacac
     7621 cctgtactcc acccggaagg acgacaaggg caacaccctg atcgtgaaca atctgaacgg
     7681 cctgtacgac aaggacaatg acaagctgaa aaagctgatc aacaagagcc ccgaaaagct
     7741 gctgatgtac caccacgacc cccagaccta ccagaaactg aagctgatta tggaacagta
     7801 cggcgacgag aagaatcccc tgtacaagta ctacgaggaa accgggaact acctgaccaa
     7861 gtactccaaa aaggacaacg gccccgtgat caagaagatt aagtattacg gcaacaaact
     7921 gaacgcccat ctggacatca ccgacgacta ccccaacagc agaaacaagg tcgtgaagct
     7981 gtccctgaag ccctacagat tcgacgtgta cctggacaat ggcgtgtaca agttcgtgac
     8041 cgtgaagaat ctggatgtga tcaaaaaaga aaactactac gaagtgaata gcaagtgcta
     8101 tgaggaagct aagaagctga agaagatcag caaccaggcc gagtttatcg cctccttcta
     8161 caacaacgat ctgatcaaga tcaacggcga gctgtataga gtgatcggcg tgaacaacga
     8221 cctgctgaac cggatcgaag tgaacatgat cgacatcacc taccgcgagt acctggaaaa
     8281 catgaacgac aagaggcccc ccaggatcat taagacaatc gcctccaaga cccagagcat
     8341 taagaagtac agcacagaca ttctgggcaa cctgtatgaa gtgaaatcta agaagcaccc
     8401 tcagatcatc aaaaagggca aaaggccggc ggccacgaaa aaggccggcc aggcaaaaaa
     8461 gaaaaaggga tcctacccat acgatgttcc agattacgct tacccatacg atgttccaga
     8521 ttacgcttac ccatacgatg ttccagatta cgctggttcg atggtgagca agggcgagga
     8581 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     8641 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     8701 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccttcggcta
     8761 cggcctgcag tgcttcgccc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     8821 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     8881 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     8941 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     9001 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     9061 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     9121 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagct accagtccgc
     9181 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     9241 cgccgggatc actctcggca tggacgagct gtacaagtga gcttgtcaag cagatcgttc
     9301 aaacatttgg caataaagtt tcttaagatt gaatcctgtt gccggtcttg cgatgattat
     9361 catataattt ctgttgaatt acgttaagca tgtaataatt aacatgtaat gcatgacgtt
     9421 atttatgaga tgggttttta tgattagagt cccgcaatta tacatttaat acgcgataga
     9481 aaacaaaata tagcgcgcaa actaggataa attatcgcgc gcggtgtcat ctatgttact
     9541 agatcgacgc t
//
