LOCUS       sequence-486463-        8585 bp    DNA     circular SYN 16-SEP-2025
DEFINITION  Donor I vector in DASH and alpha2 vector in GoldenBraid/DASH, with 
            SacB counter-selection, with attBTT and attBCC-FRT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8585)
  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 8585)
  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-DIS-alpha-2
FEATURES             Location/Qualifiers
     source          1..8585
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1..25
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     protein_bind    48..81
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     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(755..788)
                     /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    795..819
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             2120..2749
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             3178..4251
                     /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      4317..4511
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(4747..4769)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4855..4995
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5010..5027)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5181..5769)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5261..5280)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7293..8084,8085..8153))
                     /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     7916..7935
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tggcaggata tattgtggtg taaacataac aagcttccgc ggtgcgggtg ccagggcgtg
       61 cccttgggct ccccgggcgc gtactcccgt ctcagtcagg agagagaccc 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 gctggggtct ctcgcttgag acgccgcggt gcgggtgcca
      721 gggcgtgccc ccgggctccc cgggcgcgta ctccgaagtt cctatacttt ctagagaata
      781 ggaacttcaa gctttgacag gatatattgg cgggtaaacc taagagaaaa gagcgtttat
      841 tagaataatc ggatatttaa aagggcgtga aaaggtttat ccgttcgtcc atttgtatgt
      901 gcatgccaac cacagggttc ccctcgggat caaagtactt tgatccaacc cctccgctgc
      961 tatagtgcag tcggcttctg acgttcagtg cagccgtcat ctgaaaacga catgtcgcac
     1021 aagtcctaag ttacgcgaca ggctgccgcc ctgccctttt cctggcgttt tcttgtcgcg
     1081 tgttttagtc gcataaagta gaatacttgc gactagaacc ggagacatta cgccatgaac
     1141 aagagcgccg ccgctggcct gctgggctat gcccgcgtca gcaccgacga ccaggacttg
     1201 accaaccaac gggccgaact gcacgcggcc ggctgcacca agctgttttc cgagaagatc
     1261 accggcacca ggcgcgaccg cccggagctg gccaggatgc ttgaccacct acgccctggc
     1321 gacgttgtga cagtgaccag gctagaccgc ctggcccgca gcacccgcga cctactggac
     1381 attgccgagc gcatccagga ggccggcgcg ggcctgcgta gcctggcaga gccgtgggcc
     1441 gacaccacca cgccggccgg ccgcatggtg ttgaccgtgt tcgccggcat tgccgagttc
     1501 gagcgttccc taatcatcga ccgcacccgg agcgggcgcg aggccgccaa ggcccgaggc
     1561 gtgaagtttg gcccccgccc taccctcacc ccggcacaga tcgcgcacgc ccgcgagctg
     1621 atcgaccagg aaggccgcac cgtgaaagag gcggctgcac tgcttggcgt gcatcgctcg
     1681 accctgtacc gcgcacttga gcgcagcgag gaagtgacgc ccaccgaggc caggcggcgc
     1741 ggtgccttcc gtgaggacgc attgaccgag gccgacgccc tggcggccgc cgagaatgaa
     1801 cgccaagagg aacaagcatg aaaccgcacc aggacggcca ggacgaaccg tttttcatta
     1861 ccgaagagat cgaggcggag atgatcgcgg ccgggtacgt gttcgagccg cccgcgcacc
     1921 tctcaaccgt gcggctgcat gaaatcctgg ccggtttgtc tgatgccaag ctggcggcct
     1981 ggccggccag cttggccgct gaagaaaccg agcgccgccg tctaaaaagg tgatgtgtat
     2041 ttgagtaaaa cagcttgcgt catgcggtcg ctgcgtatat gatgcgatga gtaaataaac
     2101 aaatacgcaa ggggaacgca tgaaggttat cgctgtactt aaccagaaag gcgggtcagg
     2161 caagacgacc atcgcaaccc atctagcccg cgccctgcaa ctcgccgggg ccgatgttct
     2221 gttagtcgat tccgatcccc agggcagtgc ccgcgattgg gcggccgtgc gggaagatca
     2281 accgctaacc gttgtcggca tcgaccgccc gacgattgac cgcgacgtga aggccatcgg
     2341 ccggcgcgac ttcgtagtga tcgacggagc gccccaggcg gcggacttgg ctgtgtccgc
     2401 gatcaaggca gccgacttcg tgctgattcc ggtgcagcca agcccttacg acatatgggc
     2461 caccgccgac ctggtggagc tggttaagca gcgcattgag gtcacggatg gaaggctaca
     2521 agcggccttt gtcgtgtcgc gggcgatcaa aggcacgcgc atcggcggtg aggttgccga
     2581 ggcgctggcc gggtacgagc tgcccattct tgagtcccgt atcacgcagc gcgtgagcta
     2641 cccaggcact gccgccgccg gcacaaccgt tcttgaatca gaacccgagg gcgacgctgc
     2701 ccgcgaggtc caggcgctgg ccgctgaaat taaatcaaaa ctcatttgag ttaatgaggt
     2761 aaagagaaaa tgagcaaaag cacaaacacg ctaagtgccg gccgtccgag cgcacgcagc
     2821 agcaaggctg caacgttggc cagcctggca gacacgccag ccatgaagcg ggtcaacttt
     2881 cagttgccgg cggaggatca caccaagctg aagatgtacg cggtacgcca aggcaagacc
     2941 attaccgagc tgctatctga atacatcgcg cagctaccag agtaaatgag caaatgaata
     3001 aatgagtaga tgaattttag cggctaaagg aggcggcatg gaaaatcaag aacaaccagg
     3061 caccgacgcc gtggaatgcc ccatgtgtgg aggaacgggc ggttggccag gcgtaagcgg
     3121 ctgggttgcc tgccggccct gcaatggcac tggaaccccc aagcccgagg aatcggcgtg
     3181 agcggtcgca aaccatccgg cccggtacaa atcggcgcgg cgctgggtga tgacctggtg
     3241 gagaagttga aggccgcgca ggccgcccag cggcaacgca tcgaggcaga agcacgcccc
     3301 ggtgaatcgt ggcaagcggc cgctgatcga atccgcaaag aatcccggca accgccggca
     3361 gccggtgcgc cgtcgattag gaagccgccc aagggcgacg agcaaccaga ttttttcgtt
     3421 ccgatgctct atgacgtggg cacccgcgat agtcgcagca tcatggacgt ggccgttttc
     3481 cgtctgtcga agcgtgaccg acgagctggc gaggtgatcc gctacgagct tccagacggg
     3541 cacgtagagg tttccgcagg gccggccggc atggccagtg tgtgggatta cgacctggta
     3601 ctgatggcgg tttcccatct aaccgaatcc atgaaccgat accgggaagg gaagggagac
     3661 aagcccggcc gcgtgttccg tccacacgtt gcggacgtac tcaagttctg ccggcgagcc
     3721 gatggcggaa agcagaaaga cgacctggta gaaacctgca ttcggttaaa caccacgcac
     3781 gttgccatgc agcgtacgaa gaaggccaag aacggccgcc tggtgacggt atccgagggt
     3841 gaagccttga ttagccgcta caagatcgta aagagcgaaa ccgggcggcc ggagtacatc
     3901 gagatcgagc tagctgattg gatgtaccgc gagatcacag aaggcaagaa cccggacgtg
     3961 ctgacggttc accccgatta ctttttgatc gatcccggca tcggccgttt tctctaccgc
     4021 ctggcacgcc gcgccgcagg caaggcagaa gccagatggt tgttcaagac gatctacgaa
     4081 cgcagtggca gcgccggaga gttcaagaag ttctgtttca ccgtgcgcaa gctgatcggg
     4141 tcaaatgacc tgccggagta cgatttgaag gaggaggcgg ggcaggctgg cccgatccta
     4201 gtcatgcgct accgcaacct gatcgagggc gaagcatccg ccggttccta atgtacggag
     4261 cagatgctag ggcaaattgc cctagcaggg gaaaaaggtc gaaaaggact ctttcctgtg
     4321 gatagcacgt acattgggaa cccaaagccg tacattggga accggaaccc gtacattggg
     4381 aacccaaagc cgtacattgg gaaccggtca cacatgtaag tgactgatat aaaagagaaa
     4441 aaaggcgatt tttccgccta aaactcttta aaacttatta aaactcttaa aacccgcctg
     4501 gcctgtgcat aactgtctgg ccagcgcaca gccgaagagc tgcaaaaagc gcctaccctt
     4561 cggtcgctgc gctccctacg ccccgccgct tcgcgtcggc ctatcgcggc cgctggccgc
     4621 tcaaaaatgg ctggcctacg gccaggcaat ctaccagggc gcggacaagc cgcgccgtcg
     4681 ccactcgacc gccggcgccc acatcaaggc accctgcctc gcgcgtttcg gtgatgacgg
     4741 tgaaaacctc tgacacatgc agctcccggt gacggtcaca gcttgtctgt aagcggatgc
     4801 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc
     4861 catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc ggcatcagag
     4921 cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga
     4981 aaataccgca tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     5041 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     5101 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     5161 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     5221 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5281 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5341 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatcccagt
     5401 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5461 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5521 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5581 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     5641 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5701 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     5761 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     5821 tcacgttaag ggattttggt catgcattct aggtgattag ttgactgtca gctgtccttg
     5881 ctccaggatg ctgtttttga caacggatgt tttattgcct ttgatgttca ttaagaagct
     5941 tggcgcaaat gttgcctttt tatcctcgaa gaagcctctg tttgtcatgt agcttgtgat
     6001 aaccacattg ttgcctttgg cttgcggcac tgcgaagtga gagtaagtga atgtcacatc
     6061 gtttggatca agacccattt gcagcacaag ccctgttttg ttcagcggct tgtaagggcc
     6121 ggttaaagag tttgatacat aaccaagcat gtaaatatcg tttgagttaa taccatcgat
     6181 cgtcattttt gaaccgcgtg aatcagtgaa caagtaccat ttgccgttca ttttgaaaac
     6241 attcgcgcgc tcgatttcat cagttaccgt gtttgaagtg atcagcggct tcattacttt
     6301 tttcaatgtg taatcattat ttaactctat gataccgagg gcgccgttcg ctaactcagc
     6361 atcgcgtttt ttagcgctct gctgaagctt ctggctttct ttacggaaga agttcgtgcc
     6421 gccgccgtag tacgctttgt taaataaaga ttcttcgcct tggtatccgt tttctgttcc
     6481 cgtgttggct tcgaatacaa ggtatttatg gcctttgtct tcaacgtagt gaggatctct
     6541 cagcgtatgg ttgtcgccgg atgtataatt gccttcatcg ataaactgct gaacgttctg
     6601 atatgttttt ccatctccgt caaaaatcgt tttgtgatct tccactccgt tgattttgag
     6661 tgtgtcatca gattttgaca catttacctg cgctgttgtc aggctttgtt tgccgtaatg
     6721 tttaccggaa tagtcagtgt agaataaacg gatttttccg tcagatgtaa aggttgcaga
     6781 accggaccat tcttgcgtct gatctttcag gatcggatcg ttggcgtcga acttatcgct
     6841 gtctttaaag acacggcccg cgtttttcca gctgtcgatt gagttgtcgc cgaccttttg
     6901 ataaaacatg tagattgatg tgtcatcagc gtctttcggg cttcccgcaa gagcaaacac
     6961 aacgtgatag ccgttgtatt cagctactgt tccgtcagcg ttttgcagcg gccagctgtc
     7021 ccacacatca agtccttttg cagactcaat atttttaatc gttgattgat cgaattgagg
     7081 cacttggtat ttttcgtttt gctgctgttt agggatctgc agcatatcat ggcgtgtaat
     7141 atgagaaacg ccgtacgttt ctttgtatgc tttttggtta ttttctttcg cgaaggcttg
     7201 agtcgctcct cctgccagaa gtgcagtcgt aaaagtcaga actgtggctt gttttacaat
     7261 ttttttgatg ttcatgttca tgattgtcct ccttaccaat gcttaatcag tgaggcacct
     7321 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     7381 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgggaccca
     7441 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     7501 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     7561 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     7621 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     7681 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     7741 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     7801 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     7861 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     7921 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     7981 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     8041 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     8101 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact cataacagaa
     8161 cttattattt ccttcctctt ttctacagta tttaaagata ccccaagaag ctaattataa
     8221 caagacgaac tccaattcac tgttccttgc attctaaaac cttaaatacc agaaaacagc
     8281 tttttcaaag ttgttttcaa agttggcgta taacatagta tcgacggagc cgattttgaa
     8341 accgcggtga tcacaggcag caacgctctg tcatcgttac aatcaacatg ctaccctccg
     8401 cgagatcatc cgtgtttcaa acccggcagc ttagttgccg ttcttccgaa tagcatcggt
     8461 aacatgagca aagtctgccg ccttacaacg gctctcccgc tgacgccgtc ccggactgat
     8521 gggctgcctg tatcgagtgg tgattttgtg ccgagctgcc ggtcggggag ctgttggctg
     8581 gctgg
//
