LOCUS       sequence-438339-        4462 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Allows users to clone two Spacer sequences into an arrray of 
            AtU6-promoter driven sgRNAs, MoClo compatible.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4462)
  TITLE     MoClo-compatible AtU6-driven sgRNA array for plant genome 
            engineering
REFERENCE   2  (bases 1 to 4462)
  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: pJ1-g1g2-K2
FEATURES             Location/Qualifiers
     source          1..4462
                     /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"
     protein_bind    1862..1883
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1898..1903,1904..1921,1922..1928)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1936..1952
                     /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     1941..1963
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1960..1976
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1960..1976
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1972..2319
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGASVSVS
                     ELCWKQHSKLK"
     primer_bind     1995..2015
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1997..2015
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2062..2081)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2063..2081)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2091..2108)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2091..2107)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2100..2122)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2395..2413)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2481..2585
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2807..3598
                     /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      3769..4357
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4258..4277
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
       61 agcgaggaag cggtctcagg aggcccattt 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 gatggaaagc gggcagtgag cgcaacgcaa
     1861 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
     1921 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
     1981 attacgccaa gcgcgcaatt aaccctcact aaagggaaca aaagctggag gacctcgagg
     2041 gggggcccgg tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc
     2101 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     2161 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     2221 cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatc cgtctcagtt
     2281 tcagagctat gctggaaaca gcatagcaag ttgaaataag gctagtccgt tatcaacttg
     2341 aaaaagtggc accgagtcgg tgcttttttt taagcagaga ccgtggacga aagggcctcg
     2401 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     2461 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     2521 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     2581 agagtatgcg ctcacgcaac tggtccagaa ccttgaccga acgcagcggt ggtaacggcg
     2641 cagtggcggt tttcatggct tgttatgact gttttttttt ggggtacagt ctatgcctcg
     2701 ggcatccaag cagcaagcgc gttacgccgt gggtcgatgt ttgatgttat ggagcagcaa
     2761 cgatgttacg cagcagggca gtcgccctaa aacaaagtta aacatcatga gggaagcggt
     2821 gatcgccgaa gtatcgactc aactatcaga ggtagttggc gtcatcgagc gccatctcga
     2881 accgacgttg ctggccgtac atttgtacgg ctccgcagtg gatggcggcc tgaagccaca
     2941 cagcgatatt gatttgctgg ttacggtgac cgtaaggctt gatgaaacaa cgcggcgagc
     3001 tttgatcaac gaccttttgg aaacttcggc ttcccctgga gagagcgaga ttctccgcgc
     3061 tgtagaagtc accattgttg tgcacgacga catcattccg tggcgttatc cagctaagcg
     3121 cgaactgcaa tttggagaat ggcagcgcaa tgacattctt gcaggtatct tcgagccagc
     3181 cacgatcgac attgatctgg ctatcttgct gacaaaagca agagaacata gcgttgcctt
     3241 ggtaggtcca gcggcggagg aactctttga tccggttcct gaacaggatc tatttgaggc
     3301 gctaaatgaa accttaacgc tatggaactc gccgcccgac tgggctggcg atgagcgaaa
     3361 tgtagtgctt acgttgtccc gcatttggta cagcgcagta accggcaaaa tcgcgccgaa
     3421 ggatgtcgct gccgactggg caatggagcg cctgccggcc cagtatcagc ccgtcatact
     3481 tgaagctaga caggcttatc ttggacaaga agaagatcgc ttggcctcgc gcgcagatca
     3541 gttggaagaa tttgtccatt acgtaaaagg cgagatcacc aaggtagtcg gcaaataact
     3601 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     3661 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     3721 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     3781 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     3841 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     3901 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     3961 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     4021 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     4081 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     4141 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     4201 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     4261 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     4321 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     4381 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     4441 ttctgtggat aaccgtatta cc
//
