LOCUS       sequence-271978-        5285 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 5285)
  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 5285)
  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..5285
                     /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    3206..3301
                     /gene="mutant version of attL"
                     /label=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(join(5211..5285,1..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 tgctacccaa 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 attatgaggg 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 ctgttcttct 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 agcgtgagct atgagaaagc 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 cgtaccgcga
     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 gggcccaata atgattttat tttgactgat agtgacctgt
     3241 tcgttgcaac aaattgatga gcaatgcttt tttataatgc caactttgta tacaaaagtt
     3301 gccccatggc gttccctcta gagagataat gagcattgca tgtctaagtt ataaaaaatt
     3361 accacatatt ttttttgtca cacttgtttg aagtgcagtt tatctatctt tatacatata
     3421 tttaaacttt actctacgaa taatataatc tatagtacta caataatatc agtgttttag
     3481 agaatcatat aaatgaacag ttagacatgg tctaaaggac aattgagtat tttgacaaca
     3541 ggactctaca gttttatctt tttagtgtgc atgtgttctc cttttttttt gcaaatagct
     3601 tcacctatat aatacttcat ccattttatt agtacatcca tttagggttt agggttaatg
     3661 gtttttatag actaattttt ttagtacatc tattttattc tattttagcc tctaaattaa
     3721 gaaaactaaa actctatttt agttttttta tttaataatt tagatataaa atagaataaa
     3781 ataaagtgac taaaaattaa acaaataccc tttaagaaat taaaaaaact aaggaaacat
     3841 ttttcttgtt tcgagtagat aatgccagcc tgttaaacgc cgtcgacgag tctaacggac
     3901 accaaccagc gaaccagcag cgtcgcgtcg ggccaagcga agcagacggc acggcatctc
     3961 tgtcgctgcc tctggacccc tctcgagagt tccgctccac cgttggactt gctccgctgt
     4021 cggcatccag aaattgcgtg gcggagcggc agacgtgagc cggcacggca ggcggcctcc
     4081 tcctcctctc acggcaccgg cagctacggg ggattccttt cccaccgctc cttcgctttc
     4141 ccttcctcgc ccgccgtaat aaatagacac cccctccaca ccctctttcc ccaacctcgt
     4201 gttgttcgga gcgcacacac acacaaccag atctccccca aatccacccg tcggcacctc
     4261 cgcttcaagg tacgccgctc gtcctccccc ccccccccct ctctaccttc tctagatcgg
     4321 cgttccggtc catggttagg gcccggtagt tctacttctg ttcatgtttg tgttagatcc
     4381 gtgtttgtgt tagatccgtg ctgctagcgt tcgtacacgg atgcgacctg tacgtcagac
     4441 acgttctgat tgctaacttg ccagtgtttc tctttgggga atcctgggat ggctctagcc
     4501 gttccgcaga cgggatcgat ttcatgattt tttttgtttc gttgcatagg gtttggtttg
     4561 cccttttcct ttatttcaat atatgccgtg cacttgtttg tcgggtcatc ttttcatgct
     4621 tttttttgtc ttggttgtga tgatgtggtc tggttgggcg gtcgttctag atcggagtag
     4681 aattaattct gtttcaaact acctggtgga tttattaatt ttggatctgt atgtgtgtgc
     4741 catacatatt catagttacg aattgaagat gatggatgga aatatcgatc taggataggt
     4801 atacatgttg atgcgggttt tactgatgca tatacagaga tgctttttgt tcgcttggtt
     4861 gtgatgatgt ggtgtggttg ggcggtcgtt cattcgttct agatcggagt agaatactgt
     4921 ttcaaactac ctggtgtatt tattaatttt ggaactgtat gtgtgtgtca tacatcttca
     4981 tagttacgag tttaagatgg atggaaatat cgatctagga taggtataca tgttgatgtg
     5041 ggttttactg atgcatatac atgatggcat atgcagcatc tattcatatg ctctaacctt
     5101 gagtacctat ctattataat aaacaagtat gttttataat tattttgatc ttgatatact
     5161 tggatgatgg catatgcagc agctatatgt ggattttttt agccctgcct tcatacgcta
     5221 tttatttgct tggtactgtt tcttttgtcg atgctcaccc tgttgtttgg tgttacttct
     5281 gcagg
//
