LOCUS       sequence-432226-        4531 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 4531)
  TITLE     MoClo-compatible AtU6-driven sgRNA array for plant genome 
            engineering
REFERENCE   2  (bases 1 to 4531)
  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: pK7-g7g8-K8
FEATURES             Location/Qualifiers
     source          1..4531
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(200..205,206..223,224..230)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             284..964
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     CDS             1284..1589
                     /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     1468..1487
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    1851..1872
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1887..1892,1893..1910,1911..1917)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1925..1941
                     /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)."
     primer_bind     1930..1952
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1949..1965
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1949..1965
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1961..2317
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGATSLSV
                     SVSELCWKQHSKLK"
     primer_bind     1984..2004
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1986..2004
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2051..2070)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2052..2070)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2080..2097)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2080..2096)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2089..2111)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2393..2411)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2479..2583
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2805..3596
                     /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      3767..4355
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4256..4275
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4509..4526
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ggtctcaact cgcccattta agttgaaaac aatcttcaaa agtcccacat cgcttagata
       61 agaaaacgaa gctgagttta tatacagcta gagtcgaagt agtgattgaa aagcaggtgt
      121 ctagtgttat attccccaga acatcaggtt aatggggtgc ctaatgcggc cactagtggc
      181 cgcattaggc accccaggct ttacacttta tgcttccggc tcgtataatg tgtggatttt
      241 gagttaggat ccgtcgagat tttcaggagc taaggaagct aaaatggaga aaaaaatcac
      301 tggatatacc accgttgata tatcccaatg gcatcgtaaa gaacattttg aggcatttca
      361 gtcagtcgct caatgtacct ataaccagac cgttcagctg gatattacgg cctttttaaa
      421 gaccgtaaag aaaaataagc acaagtttta tccggccttt attcacattc ttgcccgcct
      481 gatgaatgct catccggaat tccgtatggc aatgaaagac ggtgagctgg tgatatggga
      541 tagtgttcac ccttgttaca ccgttttcca tgagcaaact gaaacgtttt catcgctctg
      601 gagtgaatac cacgacgatt tccggcagtt tctacacata tattcgcaag atgtggcgtg
      661 ttacggtgaa aacctggcct atttccctaa agggtttatt gagaatatgt ttttcgtttc
      721 agccaatccc tgggtgagtt tcaccagttt tgatttaaac gtggccaata tggacaactt
      781 cttcgccccc gttttcacca tgggcaaata ttatacgcaa ggcgacaagg tgctgatgcc
      841 gctggcgatt caggttcatc atgccgtttg tgatggcttc catgtcggca gaatgcttaa
      901 tgaattacaa cagtactgcg atgagtggca ggcggggcgt aatctagagg atccggctta
      961 ctaaaagcca gataacagta tgcgtatttg cgcgctgatt tttgcggtat aagaatatat
     1021 actgatatgt atacccgaag tatgtcaaaa ggaggtatgc tatgaagcag cgtattacag
     1081 tgacagttga cagcgacagc tatcagttgc tcaaggcata tatgatgtca atatctccgg
     1141 tctggtaagc acaaccatgc agaatgaagc ccgtcgtctg cgtgccgaac gctggaaagc
     1201 ggaaaatcag gaagggatgg ctgaggtcgc ccggtttatt gaaatgaacg gctcttttgc
     1261 tgacgagaac aggggctggt gaaatgcagt ttaaggttta cacctataaa agagagagcc
     1321 gttatcgtct gtttgtggat gtacagagtg atattattga cacgcccggg cgacggatgg
     1381 tgatccccct ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa ctttacccgg
     1441 tggtgcatat cggggatgaa agctggcgca tgatgaccac cgatatggcc agtgtgccgg
     1501 tttccgttat cggggaagaa gtggctgatc tcagccaccg cgaaaatgac atcaaaaacg
     1561 ccattaacct gatgttctgg ggaatataac actagacacc tgcaaaagtt tcagagctat
     1621 gctggaaaca gcatagcaag ttgaaataag gctagtccgt tatcaacttg aaaaagtggc
     1681 accgagtcgg tgcttttttt tggatccgcc catttaagtt gaaaacaatc ttcaaaagtc
     1741 ccacatcgct tagataagaa aacgaagctg agtttatata cagctagagt cgaagtagtg
     1801 attgagagac ggataaacta gtggaaagcg ggcagtgagc gcaacgcaat taatgtgagt
     1861 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt
     1921 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag
     1981 cgcgcaatta accctcacta aagggaacaa aagctggagg acctcgaggg ggggcccggt
     2041 acccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg
     2101 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     2161 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     2221 cctgaatggc gaatgggacg cgccctgtag cggcgccact agtttatccg tctcagtttc
     2281 agagctatgc tggaaacagc atagcaagtt gaaataaggc tagtccgtta tcaacttgaa
     2341 aaagtggcac cgagtcggtg cttttttttt gggagagacc gtggacgaaa gggcctcgtg
     2401 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     2461 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     2521 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     2581 agtatgcgct cacgcaactg gtccagaacc ttgaccgaac gcagcggtgg taacggcgca
     2641 gtggcggttt tcatggcttg ttatgactgt ttttttttgg ggtacagtct atgcctcggg
     2701 catccaagca gcaagcgcgt tacgccgtgg gtcgatgttt gatgttatgg agcagcaacg
     2761 atgttacgca gcagggcagt cgccctaaaa caaagttaaa catcatgagg gaagcggtga
     2821 tcgccgaagt atcgactcaa ctatcagagg tagttggcgt catcgagcgc catctcgaac
     2881 cgacgttgct ggccgtacat ttgtacggct ccgcagtgga tggcggcctg aagccacaca
     2941 gcgatattga tttgctggtt acggtgaccg taaggcttga tgaaacaacg cggcgagctt
     3001 tgatcaacga ccttttggaa acttcggctt cccctggaga gagcgagatt ctccgcgctg
     3061 tagaagtcac cattgttgtg cacgacgaca tcattccgtg gcgttatcca gctaagcgcg
     3121 aactgcaatt tggagaatgg cagcgcaatg acattcttgc aggtatcttc gagccagcca
     3181 cgatcgacat tgatctggct atcttgctga caaaagcaag agaacatagc gttgccttgg
     3241 taggtccagc ggcggaggaa ctctttgatc cggttcctga acaggatcta tttgaggcgc
     3301 taaatgaaac cttaacgcta tggaactcgc cgcccgactg ggctggcgat gagcgaaatg
     3361 tagtgcttac gttgtcccgc atttggtaca gcgcagtaac cggcaaaatc gcgccgaagg
     3421 atgtcgctgc cgactgggca atggagcgcc tgccggccca gtatcagccc gtcatacttg
     3481 aagctagaca ggcttatctt ggacaagaag aagatcgctt ggcctcgcgc gcagatcagt
     3541 tggaagaatt tgtccattac gtaaaaggcg agatcaccaa ggtagtcggc aaataactgt
     3601 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     3661 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     3721 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     3781 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     3841 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     3901 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     3961 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     4021 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4081 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     4141 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     4201 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     4261 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     4321 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     4381 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     4441 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     4501 ccgagcgcag cgagtcagtg agcgaggaag c
//
