LOCUS       sequence-432228-        4477 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Allows users to clone two Spacer sequences into an arrray of 
            AtU6-promoter driven sgRNAs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4477)
  TITLE     MoClo-compatible AtU6-driven sgRNA array for plant genome 
            engineering
REFERENCE   2  (bases 1 to 4477)
  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"
COMMENT     Sequence Label: pK3-g3g4-K4
FEATURES             Location/Qualifiers
     source          1..4477
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     49..66
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(219..224,225..242,243..249)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             303..983
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     CDS             1303..1608
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1487..1506
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1793..2137)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGATSYPS
                     LNHYFDSSCI"
     primer_bind     1987..2009
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2001..2018
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2002..2018
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2028..2047
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2028..2046
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2094..2114)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2094..2112)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2133..2149)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2133..2149)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2146..2168)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2157..2173
                     /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(2181..2187,2188..2205,2206..2211))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2226..2247
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2410..2428)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2496..2600
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2822..3613
                     /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      3784..4372
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4273..4292
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
       61 agcgaggaag cggtctcagc acgcccattt aagttgaaaa caatcttcaa aagtcccaca
      121 tcgcttagat aagaaaacga agctgagttt atatacagct agagtcgaag tagtgattga
      181 aaagcaggtg tctagtggcc gcattaggca ccccaggctt tacactttat gcttccggct
      241 cgtataatgt gtggattttg agttaggatc cgtcgagatt ttcaggagct aaggaagcta
      301 aaatggagaa aaaaatcact ggatatacca ccgttgatat atcccaatgg catcgtaaag
      361 aacattttga ggcatttcag tcagtcgctc aatgtaccta taaccagacc gttcagctgg
      421 atattacggc ctttttaaag accgtaaaga aaaataagca caagttttat ccggccttta
      481 ttcacattct tgcccgcctg atgaatgctc atccggaatt ccgtatggca atgaaagacg
      541 gtgagctggt gatatgggat agtgttcacc cttgttacac cgttttccat gagcaaactg
      601 aaacgttttc atcgctctgg agtgaatacc acgacgattt ccggcagttt ctacacatat
      661 attcgcaaga tgtggcgtgt tacggtgaaa acctggccta tttccctaaa gggtttattg
      721 agaatatgtt tttcgtttca gccaatccct gggtgagttt caccagtttt gatttaaacg
      781 tggccaatat ggacaacttc ttcgcccccg ttttcaccat gggcaaatat tatacgcaag
      841 gcgacaaggt gctgatgccg ctggcgattc aggttcatca tgccgtttgt gatggcttcc
      901 atgtcggcag aatgcttaat gaattacaac agtactgcga tgagtggcag gcggggcgta
      961 atctagagga tccggcttac taaaagccag ataacagtat gcgtatttgc gcgctgattt
     1021 ttgcggtata agaatatata ctgatatgta tacccgaagt atgtcaaaag gaggtatgct
     1081 atgaagcagc gtattacagt gacagttgac agcgacagct atcagttgct caaggcatat
     1141 atgatgtcaa tatctccggt ctggtaagca caaccatgca gaatgaagcc cgtcgtctgc
     1201 gtgccgaacg ctggaaagcg gaaaatcagg aagggatggc tgaggtcgcc cggtttattg
     1261 aaatgaacgg ctcttttgct gacgagaaca ggggctggtg aaatgcagtt taaggtttac
     1321 acctataaaa gagagagccg ttatcgtctg tttgtggatg tacagagtga tattattgac
     1381 acgcccgggc gacggatggt gatccccctg gccagtgcac gtctgctgtc agataaagtc
     1441 tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc
     1501 gatatggcca gtgtgccggt ttccgttatc ggggaagaag tggctgatct cagccaccgc
     1561 gaaaatgaca tcaaaaacgc cattaacctg atgttctggg gaatataaca ctagacacct
     1621 gcaaaagttt cagagctatg ctggaaacag catagcaagt tgaaataagg ctagtccgtt
     1681 atcaacttga aaaagtggca ccgagtcggt gctttttttt ggatccgccc atttaagttg
     1741 aaaacaatct tcaaaagtcc cacatcgctt agataagaaa acgaagctga gtttatatac
     1801 agctagagtc gaagtagtga ttgagagacg gataactagt ggcgccgcta cagggcgcgt
     1861 cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
     1921 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
     1981 ggttttccca gtcacgacgt tgtaaaacga cggccagtga gcgcgcgtaa tacgactcac
     2041 tatagggcga attgggtacc gggccccccc tcgaggtcct ccagcttttg ttccctttag
     2101 tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     2161 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     2221 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccactag
     2281 ttatccgtct cagtttcaga gctatgctgg aaacagcata gcaagttgaa ataaggctag
     2341 tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttttccac agagaccgtg
     2401 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     2461 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     2521 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     2581 aatattgaaa aaggaagagt atgcgctcac gcaactggtc cagaaccttg accgaacgca
     2641 gcggtggtaa cggcgcagtg gcggttttca tggcttgtta tgactgtttt tttttggggt
     2701 acagtctatg cctcgggcat ccaagcagca agcgcgttac gccgtgggtc gatgtttgat
     2761 gttatggagc agcaacgatg ttacgcagca gggcagtcgc cctaaaacaa agttaaacat
     2821 catgagggaa gcggtgatcg ccgaagtatc gactcaacta tcagaggtag ttggcgtcat
     2881 cgagcgccat ctcgaaccga cgttgctggc cgtacatttg tacggctccg cagtggatgg
     2941 cggcctgaag ccacacagcg atattgattt gctggttacg gtgaccgtaa ggcttgatga
     3001 aacaacgcgg cgagctttga tcaacgacct tttggaaact tcggcttccc ctggagagag
     3061 cgagattctc cgcgctgtag aagtcaccat tgttgtgcac gacgacatca ttccgtggcg
     3121 ttatccagct aagcgcgaac tgcaatttgg agaatggcag cgcaatgaca ttcttgcagg
     3181 tatcttcgag ccagccacga tcgacattga tctggctatc ttgctgacaa aagcaagaga
     3241 acatagcgtt gccttggtag gtccagcggc ggaggaactc tttgatccgg ttcctgaaca
     3301 ggatctattt gaggcgctaa atgaaacctt aacgctatgg aactcgccgc ccgactgggc
     3361 tggcgatgag cgaaatgtag tgcttacgtt gtcccgcatt tggtacagcg cagtaaccgg
     3421 caaaatcgcg ccgaaggatg tcgctgccga ctgggcaatg gagcgcctgc cggcccagta
     3481 tcagcccgtc atacttgaag ctagacaggc ttatcttgga caagaagaag atcgcttggc
     3541 ctcgcgcgca gatcagttgg aagaatttgt ccattacgta aaaggcgaga tcaccaaggt
     3601 agtcggcaaa taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     3661 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     3721 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     3781 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     3841 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     3901 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     3961 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     4021 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     4081 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     4141 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     4201 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     4261 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     4321 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     4381 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     4441 tgcgttatcc cctgattctg tggataaccg tattacc
//
