LOCUS       sequence-378280-        9069 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To test for the repressive potential of the Transcription Block (TB)
            with the FRT sites around it..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9069)
  AUTHORS   Lloyd JPB, Ly F, Gong P, Pflueger J, Swain T, Pflueger C, Fourie E, 
            Khan MA, Kidd BN, Lister R
  TITLE     Synthetic memory circuits for stable cell reprogramming in plants.
  JOURNAL   Nat Biotechnol. 2022 Jul 4. pii: 10.1038/s41587-022-01383-2. doi: 
            10.1038/s41587-022-01383-2.
  PUBMED    35788565
REFERENCE   2  (bases 1 to 9069)
  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: "Nat 
            Biotechnol. 2022 Jul 4. pii: 10.1038/s41587-022-01383-2. doi: 
            10.1038/s41587-022-01383-2."
COMMENT     Sequence Label: LLP728_L2_pV01_0I_FRT-TB-rLUC_TCTP-fLUC
FEATURES             Location/Qualifiers
     source          1..9069
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    845..878
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     polyA_signal    879..927
                     /note="synthetic polyadenylation signal"
     misc_feature    941..1032
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     981..1000
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     protein_bind    1033..1066
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     intron          1133..1321
                     /label=IV2 intron
                     /note="modified second intron of the potato ST-LS1 gene 
                     (Vancanneyt et al., 1990)"
     polyA_signal    2349..2523
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     CDS             2911..4563
                     /codon_start=1
                     /gene="luc"
                     /product="firefly luciferase"
                     /label=luciferase
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKSKL"
     primer_bind     complement(2977..2995)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     terminator      4583..4783
                     /label=gene 7 terminator
                     /note="transcription terminator for octopine-type Ti 
                     plasmid gene 7"
     misc_feature    4847..4871
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      4959..5669
                     /label=oriV
                     /note="incP origin of replication"
     promoter        5690..5794
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5795..6589
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSLREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     rep_origin      6760..7348
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7249..7268
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             7684..8832
                     /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    8978..9002
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 gtgccgaatt cggatccgga gtcgacaaaa tttagaacga acttaattat gatctcaaat
       61 acattgatac atatctcatc tagatctagg ttatcattat gtaagaaagt tttgacgaat
      121 atggcacgac aaaatggcta gactcgatgt aattggtatc tcaactcaac attatactta
      181 taccaaacat tagttagaca aaatttaaac aactattttt tatgtatgca agagtcagca
      241 tatgtataat tgattcagaa tcgttttgac gagttcggat gtagtagtag ccattattta
      301 atgtacatac taatcgtgaa tagtgaatat gatgaaacat tgtatcttat tgtataaata
      361 tccataaaca catcatgaaa gacactttct ttcacggtct gaattaatta tgatacaatt
      421 ctaatagaaa acgaattaaa ttacgttgaa ttgtatgaaa tctaattgaa caagccaacc
      481 acgacgacga ctaacgttgc ctggattgac tcggtttaag ttaaccacta aaaaaacgga
      541 gctgtcatgt aacacgcgga tcgagcaggt cacagtcatg aagccatcaa agcaaaagaa
      601 ctaatccaag ggctgagatg attaattagt ttaaaaatta gttaacacga gggaaaaggc
      661 tgtctgacag ccaggtcacg ttatctttac ctgtggtcga aatgattcgt gtctgtcgat
      721 tttaattatt tttttgaaag gccgaaaata aagttgtaag agataaaccc gcctatataa
      781 attcatatat tttcctctcc gctttgaata ctgtattttt acaacaatta ccaacaacaa
      841 caaagaagtt cctattctct agaaagtata ggaacttcaa taaaatatct ttattttcat
      901 tacatctgtg tgttggtttt ttgtgtgaat cgatagtact aacatacgct ctccatcaaa
      961 acaaaacgaa acaaaacaaa ctagcaaaat aggctgtccc cagtgcaagt gcaggtgcca
     1021 gaacatttct ctgaagttcc tattctctag aaagtatagg aacttccaac aaacaacatt
     1081 acaattacta tttacaatta caatgacttc aaaggtgtat gatcctgagc aggtaagttt
     1141 ctgcttctac ctttgatata tatataataa ttatcattaa ttagtagtaa tataatattt
     1201 caaatatttt tttcaaaata aaagaatgta gtatatagca attgcttttc tgtagtttat
     1261 aagtgtgtat attttaattt ataacttttc taatatatga ccaaaatttg ttgatgtgca
     1321 gagaaaaaga atgataactg gtccacaatg gtgggcaaga tgtaaacaga tgaacgtgct
     1381 tgattctttc attaattact acgattcaga aaagcacgct gagaatgcag ttatctttct
     1441 tcacggaaac gctgcatctt catatttgtg gagacatgtt gtgcctcaca ttgaaccagt
     1501 ggctaggtgt attatcccag atcttatcgg aatgggaaag tctggtaaat ctggaaatgg
     1561 ttcttacaga cttttggatc attataagta cctcacagct tggttcgaac ttctcaactt
     1621 acctaagaag attattttcg ttggacacga ttggggtgct tgcttggcat tccattactc
     1681 atacgaacac caagataaga tcaaagctat agttcatgca gagagtgttg tggatgtgat
     1741 agaatcttgg gatgagtggc cagatattga agaggatatt gctttgatca agtctgaaga
     1801 gggagaaaag atggttctcg aaaataactt tttcgtggaa actatgcttc cttcaaagat
     1861 tatgagaaaa ttggaaccag aagagtttgc tgcatatctc gaacctttca aggaaaaagg
     1921 agaggttaga aggccaacac tcagttggcc tagggaaata ccattagtta agggaggtaa
     1981 acctgatgtt gtgcagatcg tgagaaatta taacgcttac cttagggcat cagatgattt
     2041 gcctaagatg ttcatcgagt cagatccagg atttttcagt aatgctatag ttgaaggtgc
     2101 aaagaaattt ccaaacaccg agttcgttaa ggtgaaagga ttgcatttct cacaagaaga
     2161 tgctcctgat gagatgggaa agtatattaa gtcttttgtt gaaagagtgc tcaaaaacga
     2221 gcagggttct cgatctgatg cttctgaagt aacgtcaaca agctcccagt ctgaggtatg
     2281 cacagtggaa acgccaggtt gtgttggttc ttaagcttct ctagctagag tcgatcgaca
     2341 agctcgagtt tctccataat aatgtgtgag tagttcccag ataagggaat tagggttcct
     2401 atagggtttc gctcatgtgt tgagcatata agaaaccctt agtatgtatt tgtatttgta
     2461 aaatacttct atcaataaaa tttctaattc ctaaaaccaa aatccagtac taaaatccag
     2521 atcgctgcaa gaattcaagc ttggagccaa cactcgaatc cccacccgtt ggaccaaacc
     2581 cggctcatta agcgtcggtt cagatttatt tcctttattt aaaaaaaagg aaagggtaaa
     2641 aaatagaaaa ttggaaacag ttaaagccca aaattgtaat ttaccgagaa ttgtaaattt
     2701 acctgaaaac cctacgctat agtttcgact ataaatacca aacttaggac ctcacttcag
     2761 aatcccctcg tcgctgcgtc tctctcccgc aaccttcgat tttcgtttat tcgcatccat
     2821 cggagagaga aaacaatcaa taagcgacct actacaaaca agtaagtaag agaaaaacca
     2881 aaagaagaag agaaacaaca agaagaagta atggaggacg ccaaaaacat aaagaaaggc
     2941 ccggcgccat tctatcctct agaggatgga accgctggag agcaactgca taaggctatg
     3001 aagagatacg ccctggttcc tggaacaatt gcttttacag atgcacatat cgaggtgaac
     3061 atcacgtacg cggaatactt cgaaatgtcc gttcggttgg cagaagctat gaaacgatat
     3121 gggctgaata caaatcacag aatcgtcgta tgcagtgaaa actctcttca attctttatg
     3181 ccggtgttgg gcgcgttatt tatcggagtt gcagttgcgc ccgcgaacga catttataat
     3241 gaacgtgaat tgctcaacag tatgaacatt tcgcagccta ccgtagtgtt tgtttccaaa
     3301 aaggggttgc aaaaaatttt gaacgtgcaa aaaaaattac caataatcca gaaaattatt
     3361 atcatggatt ctaaaacgga ttaccaggga tttcagtcga tgtacacgtt cgtcacatct
     3421 catctacctc ccggttttaa tgaatacgat tttgtaccag agtcctttga tcgtgacaaa
     3481 acaattgcac tgataatgaa ttcctctgga tctactgggt tacctaaggg tgtggccctt
     3541 ccgcatagaa ctgcctgcgt cagattctcg catgccagag atcctatttt tggcaatcaa
     3601 atcattccgg atactgcgat tttaagtgtt gttccattcc atcacggttt tggaatgttt
     3661 actacactcg gatatttgat atgtggattt cgagtcgtct taatgtatag atttgaagaa
     3721 gagctgtttt tacgatccct tcaggattac aaaattcaaa gtgcgttgct agtaccaacc
     3781 ctattttcat tcttcgccaa aagcactctg attgacaaat acgatttatc taatttacac
     3841 gaaattgctt ctgggggcgc acctctttcg aaagaagtcg gggaagcggt tgcaaaacgc
     3901 ttccatcttc cagggatacg acaaggatat gggctcactg agactacatc agctattctg
     3961 attacacccg agggggatga taaaccgggc gcggtcggta aagttgttcc attttttgaa
     4021 gcgaaggttg tggatctgga taccgggaaa acgctgggcg ttaatcagag aggcgaatta
     4081 tgtgtcagag gacctatgat tatgtccggt tatgtaaaca atccggaagc gaccaacgcc
     4141 ttgattgaca aggatggatg gctacattct ggagacatag cttactggga cgaggacgaa
     4201 cacttcttca tagttgaccg cttgaagtct ttaattaaat acaaaggata tcaggtggcc
     4261 cccgctgaat tggaatcgat attgttacaa caccccaaca tcttcgacgc gggcgtggca
     4321 ggtctgcccg acgatgacgc cggtgaactt cccgccgccg ttgttgtttt ggagcacgga
     4381 aagacgatga cggaaaaaga gatcgtggat tacgtcgcca gtcaagtaac aaccgcgaaa
     4441 aagttgcgcg gaggagttgt gtttgtggac gaagtaccga aaggtcttac cggaaaactc
     4501 gacgcaagaa aaatcagaga gatcctcata aaggccaaga agggcggaaa gtccaaattg
     4561 taagcttgat cccccgtcga cagctagcta tatcatcaat ttatgtatta cacataatat
     4621 cgcactcagt ctttcatcta cggcaatgta ccagctgata taatcagtta ttgaaatatt
     4681 tctgaattta aacttgcatc aataaattta tgtttttgct tggactataa tacctgactt
     4741 gttattttat caataaatat ttaaactata tttctttcaa gatcgctact agaggatgca
     4801 catgtgaccg agggacacga agtgatccgt ttaaactatc agtgtttgac aggatatatt
     4861 ggcgggtaaa cctaagagaa aagagcgttt attagaataa tcggatattt aaaagggcgt
     4921 gaaaaggttt atccgttcgt ccatttgtat gtgccagccg cctttgcgac gctcaccggg
     4981 ctggttgccc tcgccgctgg gctggcggcc gtctatggcc ctgcaaacgc gccagaaacg
     5041 ccgtcgaagc cgtgtgcgag acaccgcggc cgccggcgtt gtggatacct cgcggaaaac
     5101 ttggccctca ctgacagatg aggggcggac gttgacactt gaggggccga ctcacccggc
     5161 gcggcgttga cagatgaggg gcaggctcga tttcggccgg cgacgtggag ctggccagcc
     5221 tcgcaaatcg gcgaaaacgc ctgattttac gcgagtttcc cacagatgat gtggacaagc
     5281 ctggggataa gtgccctgcg gtattgacac ttgaggggcg cgactactga cagatgaggg
     5341 gcgcgatcct tgacacttga ggggcagagt gctgacagat gaggggcgca cctattgaca
     5401 tttgaggggc tgtccacagg cagaaaatcc agcatttgca agggtttccg cccgtttttc
     5461 ggccaccgct aacctgtctt ttaacctgct tttaaaccaa tatttataaa ccttgttttt
     5521 aaccagggct gcgccctgtg cgcgtgaccg cgcacgccga aggggggtgc ccccccttct
     5581 cgaaccctcc cggcccgcta acgcgggcct cccatccccc caggggctgc gcccctcggc
     5641 cgcgaacggc ctcaccccaa aaatggcagc gctggccaat tcccgagtgc gcggaacccc
     5701 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     5761 ataaatgctt caataatatt gaaaaaggaa gagtatggct aaaatgagaa tatcaccgga
     5821 attgaaaaaa ctgatcgaaa aataccgctg cgtaaaagat acggaaggaa tgtctcctgc
     5881 taaggtatat aagctggtgg gagaaaatga aaacctatat ttaaaaatga cggacagccg
     5941 gtataaaggg accacctatg atgtggaacg ggaaaaggac atgatgctat ggctggaagg
     6001 aaagctgcct gttccaaagg tcctgcactt tgaacggcat gatggctgga gcaatctgct
     6061 catgagtgag gccgatggcg tcctttgctc ggaagagtat gaagatgaac aaagccctga
     6121 aaagattatc gagctgtatg cggagtgcat caggctcttt cactccatcg acatatcgga
     6181 ttgtccctat acgaatagct tagacagccg cttagccgaa ttggattact tactgaataa
     6241 cgatctggcc gatgtggatt gcgaaaactg ggaagaggac actccattta aagatccgcg
     6301 cgagctgtat gattttttaa agacggaaaa gcccgaagag gaacttgtct tttcccacgg
     6361 cgacctggga gacagcaaca tctttgtgaa agatggcaaa gtaagtggct ttattgatct
     6421 tgggagaagc ggcagggcgg acaagtggta tgacattgcc ttctgcgtcc ggtcgctcag
     6481 ggaggatatc ggggaagaac agtatgtcga gctatttttt gacttactgg ggatcaagcc
     6541 tgattgggag aaaataaaat attatatttt actggatgaa ttgttttagc tgtcagacca
     6601 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     6661 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     6721 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     6781 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     6841 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     6901 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     6961 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     7021 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     7081 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     7141 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     7201 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     7261 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     7321 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     7381 ggcagatcct agatgtggcg caacgatgcc ggcgacaagc aggagcgcac cgacttcttc
     7441 cgcatcaagt gttttggctc tcaggccgag gcccacggca agtatttggg caaggggtcg
     7501 ctggtattcg tgcagggcaa gattcggaat accaagtacg agaaggacgg ccagacggtc
     7561 tacgggaccg acttcattgc cgataaggtg gattatctgg acaccaaggc accaggcggg
     7621 tcaaatcagg aataagggca cattgccccg gcgtgagtcg gggcaatccc gcaaggaggg
     7681 tgaatgaatc ggacgtttga ccggaaggca tacaggcaag aactgatcga cgcggggttt
     7741 tccgccgagg atgccgaaac catcgcaagc cgcaccgtca tgcgtgcgcc ccgcgaaacc
     7801 ttccagtccg tcggctcgat ggtccagcaa gctacggcca agatcgagcg cgacagcgtg
     7861 caactggctc cccctgccct gcccgcgcca tcggccgccg tggagcgttc gcgtcgtctt
     7921 gaacaggagg cggcaggttt ggcgaagtcg atgaccatcg acacgcgagg aactatgacg
     7981 accaagaagc gaaaaaccgc cggcgaggac ctggcaaaac aggtcagcga ggccaagcag
     8041 gccgcgttgc tgaaacacac gaagcagcag atcaaggaaa tgcagctttc cttgttcgat
     8101 attgcgccgt ggccggacac gatgcgagcg atgccaaacg acacggcccg ctctgccctg
     8161 ttcaccacgc gcaacaagaa aatcccgcgc gaggcgctgc aaaacaaggt cattttccac
     8221 gtcaacaagg acgtgaagat cacctacacc ggcgtcgagc tgcgggccga cgatgacgaa
     8281 ctggtgtggc agcaggtgtt ggagtacgcg aagcgcaccc ctatcggcga gccgatcacc
     8341 ttcacgttct acgagctttg ccaggacctg ggctggtcga tcaatggccg gtattacacg
     8401 aaggccgagg aatgcctgtc gcgcctacag gcgacggcga tgggcttcac gtccgaccgc
     8461 gttgggcacc tggaatcggt gtcgctgctg caccgcttcc gcgtcctgga ccgtggcaag
     8521 aaaacgtccc gttgccaggt cctgatcgac gaggaaatcg tcgtgctgtt tgctggcgac
     8581 cactacacga aattcatatg ggagaagtac cgcaagctgt cgccgacggc ccgacggatg
     8641 ttcgactatt tcagctcgca ccgggagccg tacccgctca agctggaaac cttccgcctc
     8701 atgtgcggat cggattccac ccgcgtgaag aagtggcgcg agcaggtcgg cgaagcctgc
     8761 gaagagttgc gaggcagcgg cctggtggaa cacgcctggg tcaatgatga cctggtgcat
     8821 tgcaaacgct agggccttgt ggggtcagtt ccggctgggg gttcagcagc ccctgctcgg
     8881 atctgttgga ccggacagta gtcatggttg atgggctgcc tgtatcgagt ggtgattttg
     8941 tgccgagctg ccggtcgggg agctgttggc tggctggtgg caggatatat tgtggtgtaa
     9001 acaaattgac gcttagacaa cttaataaca cattgcggac gtttttaatg tactggggtt
     9061 gaacactct
//
