LOCUS       sequence-486052-        7436 bp    DNA     circular SYN 16-SEP-2025
DEFINITION  Donor II vector in DASH and omega2 vector in GoldenBraid/DASH, with 
            attPCC and attPTT-FRT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7436)
  AUTHORS   Zhao C, Stepanova AN, Alonso JM
  TITLE     DASH: a versatile and high-capacity gene stacking system for plant 
            synthetic biology.
  JOURNAL   Plant Biotechnol J. 2025 Jun 10. doi: 10.1111/pbi.70179.
  PUBMED    40493021
REFERENCE   2  (bases 1 to 7436)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 16, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Plant 
            Biotechnol J. 2025 Jun 10. doi: 10.1111/pbi.70179."
COMMENT     Sequence Label: pDASH-DII-omega-2
FEATURES             Location/Qualifiers
     source          1..7436
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     415..437
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     429..446
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     430..446
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    447..503
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(516..532)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(516..532)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(529..551)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    540..556
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(564..570,571..588,589..594))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    609..630
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    complement(756..789)
                     /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)"
     misc_feature    796..820
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             2121..2750
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             3179..4252
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="MSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
                     QAAADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
                     KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
                     GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
                     ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
                     LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
                     LVMRYRNLIEGEASAGS"
     rep_origin      4318..4512
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(4748..4770)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4856..4996
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5011..5028)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5182..5770)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5262..5281)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5858..6649)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     misc_feature    join(7435..7436,1..23)
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 gcaggatata ttgtggtgta aacataacga tatcagtagt gccccaactg gggtaacctc
       61 cgagttctct cagttggggg cgtaatcggt ctcagtcagg agtgagacgc acagcttgtc
      121 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
      181 gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc
      241 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc cattcgccat
      301 tcaggctgcg caactgttgg gaagggcgat cggcgcaggc ctcttcgcta ttacgccagc
      361 tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt
      421 cacgacgttg taaaacgacg gccagtgaat tcgagctcgg tacccgggga tcctctagag
      481 tcgacctgca ggcatgcaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
      541 tgttatccgc tcacaattcc acacaacata cgagccggaa gcatatagtg taaagcctgg
      601 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
      661 tcgggaaacc tgtcgtgcca gctgcgtctc acgcttgaga ccgatagtag tgccccaact
      721 ggggtaacct ttgagttctc tcagttgggg gcgtagaagt tcctatactt tctagagaat
      781 aggaacttcg atatctgaca ggatatattg gcgggtaaac ctaagagaaa agagcgttta
      841 ttagaataat cggatattta aaagggcgtg aaaaggttta tccgttcgtc catttgtatg
      901 tgcatgccaa ccacagggtt cccctcggga tcaaagtact ttgatccaac ccctccgctg
      961 ctatagtgca gtcggcttct gacgttcagt gcagccgtca tctgaaaacg acatgtcgca
     1021 caagtcctaa gttacgcgac aggctgccgc cctgcccttt tcctggcgtt ttcttgtcgc
     1081 gtgttttagt cgcataaagt agaatacttg cgactagaac cggagacatt acgccatgaa
     1141 caagagcgcc gccgctggcc tgctgggcta tgcccgcgtc agcaccgacg accaggactt
     1201 gaccaaccaa cgggccgaac tgcacgcggc cggctgcacc aagctgtttt ccgagaagat
     1261 caccggcacc aggcgcgacc gcccggagct ggccaggatg cttgaccacc tacgccctgg
     1321 cgacgttgtg acagtgacca ggctagaccg cctggcccgc agcacccgcg acctactgga
     1381 cattgccgag cgcatccagg aggccggcgc gggcctgcgt agcctggcag agccgtgggc
     1441 cgacaccacc acgccggccg gccgcatggt gttgaccgtg ttcgccggca ttgccgagtt
     1501 cgagcgttcc ctaatcatcg accgcacccg gagcgggcgc gaggccgcca aggcccgagg
     1561 cgtgaagttt ggcccccgcc ctaccctcac cccggcacag atcgcgcacg cccgcgagct
     1621 gatcgaccag gaaggccgca ccgtgaaaga ggcggctgca ctgcttggcg tgcatcgctc
     1681 gaccctgtac cgcgcacttg agcgcagcga ggaagtgacg cccaccgagg ccaggcggcg
     1741 cggtgccttc cgtgaggacg cattgaccga ggccgacgcc ctggcggccg ccgagaatga
     1801 acgccaagag gaacaagcat gaaaccgcac caggacggcc aggacgaacc gtttttcatt
     1861 accgaagaga tcgaggcgga gatgatcgcg gccgggtacg tgttcgagcc gcccgcgcac
     1921 ctctcaaccg tgcggctgca tgaaatcctg gccggtttgt ctgatgccaa gctggcggcc
     1981 tggccggcca gcttggccgc tgaagaaacc gagcgccgcc gtctaaaaag gtgatgtgta
     2041 tttgagtaaa acagcttgcg tcatgcggtc gctgcgtata tgatgcgatg agtaaataaa
     2101 caaatacgca aggggaacgc atgaaggtta tcgctgtact taaccagaaa ggcgggtcag
     2161 gcaagacgac catcgcaacc catctagccc gcgccctgca actcgccggg gccgatgttc
     2221 tgttagtcga ttccgatccc cagggcagtg cccgcgattg ggcggccgtg cgggaagatc
     2281 aaccgctaac cgttgtcggc atcgaccgcc cgacgattga ccgcgacgtg aaggccatcg
     2341 gccggcgcga cttcgtagtg atcgacggag cgccccaggc ggcggacttg gctgtgtccg
     2401 cgatcaaggc agccgacttc gtgctgattc cggtgcagcc aagcccttac gacatatggg
     2461 ccaccgccga cctggtggag ctggttaagc agcgcattga ggtcacggat ggaaggctac
     2521 aagcggcctt tgtcgtgtcg cgggcgatca aaggcacgcg catcggcggt gaggttgccg
     2581 aggcgctggc cgggtacgag ctgcccattc ttgagtcccg tatcacgcag cgcgtgagct
     2641 acccaggcac tgccgccgcc ggcacaaccg ttcttgaatc agaacccgag ggcgacgctg
     2701 cccgcgaggt ccaggcgctg gccgctgaaa ttaaatcaaa actcatttga gttaatgagg
     2761 taaagagaaa atgagcaaaa gcacaaacac gctaagtgcc ggccgtccga gcgcacgcag
     2821 cagcaaggct gcaacgttgg ccagcctggc agacacgcca gccatgaagc gggtcaactt
     2881 tcagttgccg gcggaggatc acaccaagct gaagatgtac gcggtacgcc aaggcaagac
     2941 cattaccgag ctgctatctg aatacatcgc gcagctacca gagtaaatga gcaaatgaat
     3001 aaatgagtag atgaatttta gcggctaaag gaggcggcat ggaaaatcaa gaacaaccag
     3061 gcaccgacgc cgtggaatgc cccatgtgtg gaggaacggg cggttggcca ggcgtaagcg
     3121 gctgggttgc ctgccggccc tgcaatggca ctggaacccc caagcccgag gaatcggcgt
     3181 gagcggtcgc aaaccatccg gcccggtaca aatcggcgcg gcgctgggtg atgacctggt
     3241 ggagaagttg aaggccgcgc aggccgccca gcggcaacgc atcgaggcag aagcacgccc
     3301 cggtgaatcg tggcaagcgg ccgctgatcg aatccgcaaa gaatcccggc aaccgccggc
     3361 agccggtgcg ccgtcgatta ggaagccgcc caagggcgac gagcaaccag attttttcgt
     3421 tccgatgctc tatgacgtgg gcacccgcga tagtcgcagc atcatggacg tggccgtttt
     3481 ccgtctgtcg aagcgtgacc gacgagctgg cgaggtgatc cgctacgagc ttccagacgg
     3541 gcacgtagag gtttccgcag ggccggccgg catggccagt gtgtgggatt acgacctggt
     3601 actgatggcg gtttcccatc taaccgaatc catgaaccga taccgggaag ggaagggaga
     3661 caagcccggc cgcgtgttcc gtccacacgt tgcggacgta ctcaagttct gccggcgagc
     3721 cgatggcgga aagcagaaag acgacctggt agaaacctgc attcggttaa acaccacgca
     3781 cgttgccatg cagcgtacga agaaggccaa gaacggccgc ctggtgacgg tatccgaggg
     3841 tgaagccttg attagccgct acaagatcgt aaagagcgaa accgggcggc cggagtacat
     3901 cgagatcgag ctagctgatt ggatgtaccg cgagatcaca gaaggcaaga acccggacgt
     3961 gctgacggtt caccccgatt actttttgat cgatcccggc atcggccgtt ttctctaccg
     4021 cctggcacgc cgcgccgcag gcaaggcaga agccagatgg ttgttcaaga cgatctacga
     4081 acgcagtggc agcgccggag agttcaagaa gttctgtttc accgtgcgca agctgatcgg
     4141 gtcaaatgac ctgccggagt acgatttgaa ggaggaggcg gggcaggctg gcccgatcct
     4201 agtcatgcgc taccgcaacc tgatcgaggg cgaagcatcc gccggttcct aatgtacgga
     4261 gcagatgcta gggcaaattg ccctagcagg ggaaaaaggt cgaaaaggac tctttcctgt
     4321 ggatagcacg tacattggga acccaaagcc gtacattggg aaccggaacc cgtacattgg
     4381 gaacccaaag ccgtacattg ggaaccggtc acacatgtaa gtgactgata taaaagagaa
     4441 aaaaggcgat ttttccgcct aaaactcttt aaaacttatt aaaactctta aaacccgcct
     4501 ggcctgtgca taactgtctg gccagcgcac agccgaagag ctgcaaaaag cgcctaccct
     4561 tcggtcgctg cgctccctac gccccgccgc ttcgcgtcgg cctatcgcgg ccgctggccg
     4621 ctcaaaaatg gctggcctac ggccaggcaa tctaccaggg cgcggacaag ccgcgccgtc
     4681 gccactcgac cgccggcgcc cacatcaagg caccctgcct cgcgcgtttc ggtgatgacg
     4741 gtgaaaacct ctgacacatg cagctcccgg tgacggtcac agcttgtctg taagcggatg
     4801 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag
     4861 ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga
     4921 gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag
     4981 aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     5041 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     5101 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     5161 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     5221 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     5281 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     5341 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     5401 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     5461 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     5521 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5581 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg
     5641 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5701 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5761 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5821 ctcacgttaa gggattttgg tcatgcattc taggtgatta tttgccgact accttggtga
     5881 tctcgccttt cacgtagtgg acaaattctt ccaactgatc tgcgcgcgag gccaagcgat
     5941 cttcttcttg tccaagataa gcctgtctag cttcaagtat gacgggctga tactgggccg
     6001 gcaggcgctc cattgcccag tcggcagcga catccttcgg cgcgattttg ccggttactg
     6061 cgctgtacca aatgcgggac aacgtaagca ctacatttcg ctcatcacca gcccagtcgg
     6121 gcggcgagtt ccatagcgtt aaggtttcat ttagcgcctc aaatagatcc tgttcaggaa
     6181 ccggatcaaa gagttcctcc gccgctggac ctaccaaggc aacgctatgt tctcttgctt
     6241 ttgtcagcaa gatagccaga tcaatgtcga tcgtggctgg ctcgaagata cctgcaagaa
     6301 tgtcattgcg ctgccattct ccaaattgca gttcgcgctt agctggataa cgccacggaa
     6361 tgatgtcgtc gtgcacaaca atggtgactt ctacagcgcg gagaatctcg ctctctccag
     6421 gggaagccga agtttccaaa aggtcgttga tcaaagctcg ccgcgttgtt tcatcaagcc
     6481 ttacggtcac cgtaaccagc aaatcaatat cactgtgtgg cttcaggccg ccatccactg
     6541 cggagccgta caaatgtacg gccagcaacg tcggttcgag atggcgctcg atgacgccaa
     6601 ctacctctga tagttgagtc gatacttcgg cgatcaccgc ttccctcata atgtttaact
     6661 ttgttttagg gcgactgccc tgctgcgtaa catcgttgct gctccataac atcaaacatc
     6721 gacccacggc gtaacgcgct tgctgcttgg atgcccgagg catagactgt accccaaaaa
     6781 aacagtcata acaagccatg aaaaccgcca ctgcgccgtt accaccgctg cgttcggtca
     6841 aggttctgga ccagttgcgt gagcgcatac gctacttgca ttacagctta cgaaccgaac
     6901 aggcttatgt ccactgggtt cgtgccttca tccgtttcca cggtgtgcgt cacccggcaa
     6961 ccttgggtag cagcgaagtc gaggcatttc tgtcctggct ggaacagaac ttattatttc
     7021 cttcctcttt tctacagtat ttaaagatac cccaagaagc taattataac aagacgaact
     7081 ccaattcact gttccttgca ttctaaaacc ttaaatacca gaaaacagct ttttcaaagt
     7141 tgttttcaaa gttggcgtat aacatagtat cgacggagcc gattttgaaa ccgcggtgat
     7201 cacaggcagc aacgctctgt catcgttaca atcaacatgc taccctccgc gagatcatcc
     7261 gtgtttcaaa cccggcagct tagttgccgt tcttccgaat agcatcggta acatgagcaa
     7321 agtctgccgc cttacaacgg ctctcccgct gacgccgtcc cggactgatg ggctgcctgt
     7381 atcgagtggt gattttgtgc cgagctgccg gtcggggagc tgttggctgg ctggtg
//
