LOCUS       sequence-266596-        5286 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Golden Gate recipient and Gateway entry vector; assembly of 4 gRNAs 
            driven by Zea mays Ubi promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5286)
  AUTHORS   Zhang Y, Ren Q, Tang X, Liu S, Malzahn AA, Zhou J, Wang J, Yin D, 
            Pan C, Yuan M, Huang L, Yang H, Zhao Y, Fang Q, Zheng X, Tian L, 
            Cheng Y, Le Y, McCoy B, Franklin L, Selengut JD, Mount SM, Que Q, 
            Zhang Y, Qi Y
  TITLE     Expanding the scope of plant genome engineering with Cas12a 
            orthologs and highly multiplexable editing systems.
  JOURNAL   Nat Commun. 2021 Mar 29;12(1):1944. doi: 10.1038/s41467-021-22330-w.
  PUBMED    33782402
REFERENCE   2  (bases 1 to 5286)
  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 
            Commun."; date: "2021-03-29"; volume: "12(1):1944. doi"; pages: " 
            10.1038/s41467-021-22330-w"
COMMENT     Sequence Label: pYPQ144-ZmUbi-pT
FEATURES             Location/Qualifiers
     source          1..5286
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     189..205
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        215..233
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(281..301)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(281..299)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(320..336)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(333..355)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    344..360
                     /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(368..374,375..392,393..398))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    413..434
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    complement(517..616)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(634..652)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(657..673)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1104..1895
                     /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"
     rep_origin      1988..2576
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2477..2496
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2730..2747
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2906..2933)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3025..3111)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3175..3191
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3207..3302
                     /gene="mutant version of attL"
                     /label=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(join(5212..5286,1..324))
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGAGLDSH
                     HLGIPAEVTPNNRVSIDKRNSTKQINSV"
ORIGIN
        1 gatccccaag tggtggctat cgagaccggc gccgctacag ggcgcgtccc attcgccatt
       61 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct
      121 ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc
      181 acgacgttgt aaaacgacgg ccagtgagcg cgcgtaatac gactcactat agggcgaatt
      241 gggtaccggg ccccccctcg aggtcctcca gcttttgttc cctttagtga gggttaattg
      301 cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
      361 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
      421 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccaccggtgg tctcttgtta
      481 ctagtttttt tgctagcaga tctaagggcg aattcgaccc agctttcttg tacaaagttg
      541 gcattataaa aaataattgc tcatcaattt gttgcaacga acaggtcact atcagtcaaa
      601 ataaaatcat tatttgccat ccagctgata tcccctatag tgagtcgtat tacatggtca
      661 tagctgtttc ctggcagctc tggcccgtgt ctcaaaatct ctgatgttac attgcacaag
      721 ataaaaatat atcatcatgc ctcctctgga ccagccagga cagaaatgcc tcgacttcgc
      781 tgctgcccaa ggttgccggg tgacgcacac cgtggaaacg gatgaaggca cgaacccagt
      841 ggacataagc ctgttcggtt cgtaagctgt aatgcaagta gcgtatgcgc tcacgcaact
      901 ggtccagaac cttgaccgaa cgcagcggtg gtaacggcgc agtggcggtt ttcatggctt
      961 gttatgactg tttttttggg gtacagtcta tgcctcgggc atccaagcag caagcgcgtt
     1021 acgccgtggg tcgatgtttg atgttatgga gcagcaacga tgttacgcag cagggcagtc
     1081 gccctaaaac aaagttaaac atcatgaggg aagcggtgat cgccgaagta tcgactcaac
     1141 tatcagaggt agttggcgtc atcgagcgcc atctcgaacc gacgttgctg gccgtacatt
     1201 tgtacggctc cgcagtggat ggcggcctga agccacacag tgatattgat ttgctggtta
     1261 cggtgaccgt aaggcttgat gaaacaacgc ggcgagcttt gatcaacgac cttttggaaa
     1321 cttcggcttc ccctggagag agcgagattc tccgcgctgt agaagtcacc attgttgtgc
     1381 acgacgacat cattccgtgg cgttatccag ctaagcgcga actgcaattt ggagaatggc
     1441 agcgcaatga cattcttgca ggtatcttcg agccagccac gatcgacatt gatctggcta
     1501 tcttgctgac aaaagcaaga gaacatagcg ttgccttggt aggtccagcg gcggaggaac
     1561 tctttgatcc ggttcctgaa caggatctat ttgaggcgct aaatgaaacc ttaacgctat
     1621 ggaactcgcc gcccgactgg gctggcgatg agcgaaatgt agtgcttacg ttgtcccgca
     1681 tttggtacag cgcagtaacc ggcaaaatcg cgccgaagga tgtcgctgcc gactgggcaa
     1741 tggagcgcct gccggcccag tatcagcccg tcatacttga agctagacag gcttatcttg
     1801 gacaagaaga agatcgcttg gcctcgcgcg cagatcagtt ggaagaattt gtccactacg
     1861 tgaaaggcga gatcaccaag gtagtcggca aataaccctc gagccaccca tgaccaaaat
     1921 cccttaacgt gagttacgcg tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     1981 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     2041 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     2101 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     2161 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     2221 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     2281 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     2341 gaacgaccta caccgaactg agatacctac agcgtgagca ttgagaaagc gccacgcttc
     2401 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     2461 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     2521 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     2581 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     2641 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     2701 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     2761 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     2821 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatacg cgtaccgctc
     2881 gccaggaaga gtttgtagaa acgcaaaaag gccatccgtc aggatggcct tctgcttagt
     2941 ttgatgcctg gcagtttatg gcgggcgtcc tgcccgccac cctccgggcc gttgcttcac
     3001 aacgttcaaa tccgctcccg gcggatttgt cctactcagg agagcgttca ccgacaaaca
     3061 acagataaaa cgaaaggccc agtcttccga ctgagccttt cgttttattt gatgcctggc
     3121 agttccctac tctcgcgtta acgcttgcat ggatgttttc ccagtcacga cgttgtaaaa
     3181 cgacggccag tcttaagctc gggcccaaat aatgatttta ttttgactga tagtgacctg
     3241 ttcgttgcaa caaattgatg agcaatgctt ttttataatg ccaactttgt atacaaaagt
     3301 tgccccatgg cgttccctct agagagataa tgagcattgc atgtctaagt tataaaaaat
     3361 taccacatat tttttttgtc acacttgttt gaagtgcagt ttatctatct ttatacatat
     3421 atttaaactt tactctacga ataatataat ctatagtact acaataatat cagtgtttta
     3481 gagaatcata taaatgaaca gttagacatg gtctaaagga caattgagta ttttgacaac
     3541 aggactctac agttttatct ttttagtgtg catgtgttct cctttttttt tgcaaatagc
     3601 ttcacctata taatacttca tccattttat tagtacatcc atttagggtt tagggttaat
     3661 ggtttttata gactaatttt tttagtacat ctattttatt ctattttagc ctctaaatta
     3721 agaaaactaa aactctattt tagttttttt atttaataat ttagatataa aatagaataa
     3781 aataaagtga ctaaaaatta aacaaatacc ctttaagaaa ttaaaaaaac taaggaaaca
     3841 tttttcttgt ttcgagtaga taatgccagc ctgttaaacg ccgtcgacga gtctaacgga
     3901 caccaaccag cgaaccagca gcgtcgcgtc gggccaagcg aagcagacgg cacggcatct
     3961 ctgtcgctgc ctctggaccc ctctcgagag ttccgctcca ccgttggact tgctccgctg
     4021 tcggcatcca gaaattgcgt ggcggagcgg cagacgtgag ccggcacggc aggcggcctc
     4081 ctcctcctct cacggcaccg gcagctacgg gggattcctt tcccaccgct ccttcgcttt
     4141 cccttcctcg cccgccgtaa taaatagaca ccccctccac accctctttc cccaacctcg
     4201 tgttgttcgg agcgcacaca cacacaacca gatctccccc aaatccaccc gtcggcacct
     4261 ccgcttcaag gtacgccgct cgtcctcccc cccccccccc tctctacctt ctctagatcg
     4321 gcgttccggt ccatggttag ggcccggtag ttctacttct gttcatgttt gtgttagatc
     4381 cgtgtttgtg ttagatccgt gctgctagcg ttcgtacacg gatgcgacct gtacgtcaga
     4441 cacgttctga ttgctaactt gccagtgttt ctctttgggg aatcctggga tggctctagc
     4501 cgttccgcag acgggatcga tttcatgatt ttttttgttt cgttgcatag ggtttggttt
     4561 gcccttttcc tttatttcaa tatatgccgt gcacttgttt gtcgggtcat cttttcatgc
     4621 ttttttttgt cttggttgtg atgatgtggt ctggttgggc ggtcgttcta gatcggagta
     4681 gaattaattc tgtttcaaac tacctggtgg atttattaat tttggatctg tatgtgtgtg
     4741 ccatacatat tcatagttac gaattgaaga tgatggatgg aaatatcgat ctaggatagg
     4801 tatacatgtt gatgcgggtt ttactgatgc atatacagag atgctttttg ttcgcttggt
     4861 tgtgatgatg tggtgtggtt gggcggtcgt tcattcgttc tagatcggag tagaatactg
     4921 tttcaaacta cctggtgtat ttattaattt tggaactgta tgtgtgtgtc atacatcttc
     4981 atagttacga gtttaagatg gatggaaata tcgatctagg ataggtatac atgttgatgt
     5041 gggttttact gatgcatata catgatggca tatgcagcat ctattcatat gctctaacct
     5101 tgagtaccta tctattataa taaacaagta tgttttataa ttattttgat cttgatatac
     5161 ttggatgatg gcatatgcag cagctatatg tggatttttt tagccctgcc ttcatacgct
     5221 atttatttgc ttggtactgt ttcttttgtc gatgctcacc ctgttgtttg gtgttacttc
     5281 tgcagg
//
