LOCUS       sequence-495605-        8589 bp    DNA     circular SYN 17-DEC-2025
DEFINITION  Donor I vector in DASH and alpha2 vector in GoldenBraid/DASH, with 
            SacB counter-selection, compatible with MoClo, with attBTT and 
            attBCC-FRT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8589)
  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 8589)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 17, 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 (MoClo)
FEATURES             Location/Qualifiers
     source          1..8589
                     /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(759..792)
                     /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    799..823
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             2124..2753
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             3182..4255
                     /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      4321..4515
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(4751..4773)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4859..4999
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5014..5031)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5185..5773)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5265..5284)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7297..8088,8089..8157))
                     /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     7920..7939
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tggcaggata tattgtggtg taaacataac aagcttccgc ggtgcgggtg ccagggcgtg
       61 cccttgggct ccccgggcgc gtactcccgt ctcagtcatg ccagagaccc 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 ctgggacgct tgagacgccg cggtgcgggt
      721 gccagggcgt gcccccgggc tccccgggcg cgtactccga agttcctata ctttctagag
      781 aataggaact tcaagctttg acaggatata ttggcgggta aacctaagag aaaagagcgt
      841 ttattagaat aatcggatat ttaaaagggc gtgaaaaggt ttatccgttc gtccatttgt
      901 atgtgcatgc caaccacagg gttcccctcg ggatcaaagt actttgatcc aacccctccg
      961 ctgctatagt gcagtcggct tctgacgttc agtgcagccg tcatctgaaa acgacatgtc
     1021 gcacaagtcc taagttacgc gacaggctgc cgccctgccc ttttcctggc gttttcttgt
     1081 cgcgtgtttt agtcgcataa agtagaatac ttgcgactag aaccggagac attacgccat
     1141 gaacaagagc gccgccgctg gcctgctggg ctatgcccgc gtcagcaccg acgaccagga
     1201 cttgaccaac caacgggccg aactgcacgc ggccggctgc accaagctgt tttccgagaa
     1261 gatcaccggc accaggcgcg accgcccgga gctggccagg atgcttgacc acctacgccc
     1321 tggcgacgtt gtgacagtga ccaggctaga ccgcctggcc cgcagcaccc gcgacctact
     1381 ggacattgcc gagcgcatcc aggaggccgg cgcgggcctg cgtagcctgg cagagccgtg
     1441 ggccgacacc accacgccgg ccggccgcat ggtgttgacc gtgttcgccg gcattgccga
     1501 gttcgagcgt tccctaatca tcgaccgcac ccggagcggg cgcgaggccg ccaaggcccg
     1561 aggcgtgaag tttggccccc gccctaccct caccccggca cagatcgcgc acgcccgcga
     1621 gctgatcgac caggaaggcc gcaccgtgaa agaggcggct gcactgcttg gcgtgcatcg
     1681 ctcgaccctg taccgcgcac ttgagcgcag cgaggaagtg acgcccaccg aggccaggcg
     1741 gcgcggtgcc ttccgtgagg acgcattgac cgaggccgac gccctggcgg ccgccgagaa
     1801 tgaacgccaa gaggaacaag catgaaaccg caccaggacg gccaggacga accgtttttc
     1861 attaccgaag agatcgaggc ggagatgatc gcggccgggt acgtgttcga gccgcccgcg
     1921 cacctctcaa ccgtgcggct gcatgaaatc ctggccggtt tgtctgatgc caagctggcg
     1981 gcctggccgg ccagcttggc cgctgaagaa accgagcgcc gccgtctaaa aaggtgatgt
     2041 gtatttgagt aaaacagctt gcgtcatgcg gtcgctgcgt atatgatgcg atgagtaaat
     2101 aaacaaatac gcaaggggaa cgcatgaagg ttatcgctgt acttaaccag aaaggcgggt
     2161 caggcaagac gaccatcgca acccatctag cccgcgccct gcaactcgcc ggggccgatg
     2221 ttctgttagt cgattccgat ccccagggca gtgcccgcga ttgggcggcc gtgcgggaag
     2281 atcaaccgct aaccgttgtc ggcatcgacc gcccgacgat tgaccgcgac gtgaaggcca
     2341 tcggccggcg cgacttcgta gtgatcgacg gagcgcccca ggcggcggac ttggctgtgt
     2401 ccgcgatcaa ggcagccgac ttcgtgctga ttccggtgca gccaagccct tacgacatat
     2461 gggccaccgc cgacctggtg gagctggtta agcagcgcat tgaggtcacg gatggaaggc
     2521 tacaagcggc ctttgtcgtg tcgcgggcga tcaaaggcac gcgcatcggc ggtgaggttg
     2581 ccgaggcgct ggccgggtac gagctgccca ttcttgagtc ccgtatcacg cagcgcgtga
     2641 gctacccagg cactgccgcc gccggcacaa ccgttcttga atcagaaccc gagggcgacg
     2701 ctgcccgcga ggtccaggcg ctggccgctg aaattaaatc aaaactcatt tgagttaatg
     2761 aggtaaagag aaaatgagca aaagcacaaa cacgctaagt gccggccgtc cgagcgcacg
     2821 cagcagcaag gctgcaacgt tggccagcct ggcagacacg ccagccatga agcgggtcaa
     2881 ctttcagttg ccggcggagg atcacaccaa gctgaagatg tacgcggtac gccaaggcaa
     2941 gaccattacc gagctgctat ctgaatacat cgcgcagcta ccagagtaaa tgagcaaatg
     3001 aataaatgag tagatgaatt ttagcggcta aaggaggcgg catggaaaat caagaacaac
     3061 caggcaccga cgccgtggaa tgccccatgt gtggaggaac gggcggttgg ccaggcgtaa
     3121 gcggctgggt tgcctgccgg ccctgcaatg gcactggaac ccccaagccc gaggaatcgg
     3181 cgtgagcggt cgcaaaccat ccggcccggt acaaatcggc gcggcgctgg gtgatgacct
     3241 ggtggagaag ttgaaggccg cgcaggccgc ccagcggcaa cgcatcgagg cagaagcacg
     3301 ccccggtgaa tcgtggcaag cggccgctga tcgaatccgc aaagaatccc ggcaaccgcc
     3361 ggcagccggt gcgccgtcga ttaggaagcc gcccaagggc gacgagcaac cagatttttt
     3421 cgttccgatg ctctatgacg tgggcacccg cgatagtcgc agcatcatgg acgtggccgt
     3481 tttccgtctg tcgaagcgtg accgacgagc tggcgaggtg atccgctacg agcttccaga
     3541 cgggcacgta gaggtttccg cagggccggc cggcatggcc agtgtgtggg attacgacct
     3601 ggtactgatg gcggtttccc atctaaccga atccatgaac cgataccggg aagggaaggg
     3661 agacaagccc ggccgcgtgt tccgtccaca cgttgcggac gtactcaagt tctgccggcg
     3721 agccgatggc ggaaagcaga aagacgacct ggtagaaacc tgcattcggt taaacaccac
     3781 gcacgttgcc atgcagcgta cgaagaaggc caagaacggc cgcctggtga cggtatccga
     3841 gggtgaagcc ttgattagcc gctacaagat cgtaaagagc gaaaccgggc ggccggagta
     3901 catcgagatc gagctagctg attggatgta ccgcgagatc acagaaggca agaacccgga
     3961 cgtgctgacg gttcaccccg attacttttt gatcgatccc ggcatcggcc gttttctcta
     4021 ccgcctggca cgccgcgccg caggcaaggc agaagccaga tggttgttca agacgatcta
     4081 cgaacgcagt ggcagcgccg gagagttcaa gaagttctgt ttcaccgtgc gcaagctgat
     4141 cgggtcaaat gacctgccgg agtacgattt gaaggaggag gcggggcagg ctggcccgat
     4201 cctagtcatg cgctaccgca acctgatcga gggcgaagca tccgccggtt cctaatgtac
     4261 ggagcagatg ctagggcaaa ttgccctagc aggggaaaaa ggtcgaaaag gactctttcc
     4321 tgtggatagc acgtacattg ggaacccaaa gccgtacatt gggaaccgga acccgtacat
     4381 tgggaaccca aagccgtaca ttgggaaccg gtcacacatg taagtgactg atataaaaga
     4441 gaaaaaaggc gatttttccg cctaaaactc tttaaaactt attaaaactc ttaaaacccg
     4501 cctggcctgt gcataactgt ctggccagcg cacagccgaa gagctgcaaa aagcgcctac
     4561 ccttcggtcg ctgcgctccc tacgccccgc cgcttcgcgt cggcctatcg cggccgctgg
     4621 ccgctcaaaa atggctggcc tacggccagg caatctacca gggcgcggac aagccgcgcc
     4681 gtcgccactc gaccgccggc gcccacatca aggcaccctg cctcgcgcgt ttcggtgatg
     4741 acggtgaaaa cctctgacac atgcagctcc cggtgacggt cacagcttgt ctgtaagcgg
     4801 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg
     4861 cagccatgac ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc
     4921 agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag
     4981 gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     5041 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     5101 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     5161 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     5221 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     5281 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     5341 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatcc
     5401 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     5461 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     5521 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     5581 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat
     5641 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     5701 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     5761 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     5821 aaactcacgt taagggattt tggtcatgca ttctaggtga ttagttgact gtcagctgtc
     5881 cttgctccag gatgctgttt ttgacaacgg atgttttatt gcctttgatg ttcattaaga
     5941 agcttggcgc aaatgttgcc tttttatcct cgaagaagcc tctgtttgtc atgtagcttg
     6001 tgataaccac attgttgcct ttggcttgcg gcactgcgaa gtgagagtaa gtgaatgtca
     6061 catcgtttgg atcaagaccc atttgcagca caagccctgt tttgttcagc ggcttgtaag
     6121 ggccggttaa agagtttgat acataaccaa gcatgtaaat atcgtttgag ttaataccat
     6181 cgatcgtcat ttttgaaccg cgtgaatcag tgaacaagta ccatttgccg ttcattttga
     6241 aaacattcgc gcgctcgatt tcatcagtta ccgtgtttga agtgatcagc ggcttcatta
     6301 cttttttcaa tgtgtaatca ttatttaact ctatgatacc gagggcgccg ttcgctaact
     6361 cagcatcgcg ttttttagcg ctctgctgaa gcttctggct ttctttacgg aagaagttcg
     6421 tgccgccgcc gtagtacgct ttgttaaata aagattcttc gccttggtat ccgttttctg
     6481 ttcccgtgtt ggcttcgaat acaaggtatt tatggccttt gtcttcaacg tagtgaggat
     6541 ctctcagcgt atggttgtcg ccggatgtat aattgccttc atcgataaac tgctgaacgt
     6601 tctgatatgt ttttccatct ccgtcaaaaa tcgttttgtg atcttccact ccgttgattt
     6661 tgagtgtgtc atcagatttt gacacattta cctgcgctgt tgtcaggctt tgtttgccgt
     6721 aatgtttacc ggaatagtca gtgtagaata aacggatttt tccgtcagat gtaaaggttg
     6781 cagaaccgga ccattcttgc gtctgatctt tcaggatcgg atcgttggcg tcgaacttat
     6841 cgctgtcttt aaagacacgg cccgcgtttt tccagctgtc gattgagttg tcgccgacct
     6901 tttgataaaa catgtagatt gatgtgtcat cagcgtcttt cgggcttccc gcaagagcaa
     6961 acacaacgtg atagccgttg tattcagcta ctgttccgtc agcgttttgc agcggccagc
     7021 tgtcccacac atcaagtcct tttgcagact caatattttt aatcgttgat tgatcgaatt
     7081 gaggcacttg gtatttttcg ttttgctgct gtttagggat ctgcagcata tcatggcgtg
     7141 taatatgaga aacgccgtac gtttctttgt atgctttttg gttattttct ttcgcgaagg
     7201 cttgagtcgc tcctcctgcc agaagtgcag tcgtaaaagt cagaactgtg gcttgtttta
     7261 caattttttt gatgttcatg ttcatgattg tcctccttac caatgcttaa tcagtgaggc
     7321 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     7381 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcggga
     7441 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     7501 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     7561 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     7621 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     7681 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     7741 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     7801 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     7861 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     7921 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     7981 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     8041 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     8101 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcataac
     8161 agaacttatt atttccttcc tcttttctac agtatttaaa gataccccaa gaagctaatt
     8221 ataacaagac gaactccaat tcactgttcc ttgcattcta aaaccttaaa taccagaaaa
     8281 cagctttttc aaagttgttt tcaaagttgg cgtataacat agtatcgacg gagccgattt
     8341 tgaaaccgcg gtgatcacag gcagcaacgc tctgtcatcg ttacaatcaa catgctaccc
     8401 tccgcgagat catccgtgtt tcaaacccgg cagcttagtt gccgttcttc cgaatagcat
     8461 cggtaacatg agcaaagtct gccgccttac aacggctctc ccgctgacgc cgtcccggac
     8521 tgatgggctg cctgtatcga gtggtgattt tgtgccgagc tgccggtcgg ggagctgttg
     8581 gctggctgg
//
