LOCUS       sequence-432227-        4479 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 4479)
  TITLE     MoClo-compatible AtU6-driven sgRNA array for plant genome 
            engineering
REFERENCE   2  (bases 1 to 4479)
  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: pK5-g5g6-K6
FEATURES             Location/Qualifiers
     source          1..4479
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(148..153,154..171,172..178)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             232..912
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     CDS             1232..1537
                     /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     1416..1435
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    1799..1820
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1835..1840,1841..1858,1859..1865)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1873..1889
                     /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     1878..1900
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1897..1913
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1897..1913
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1909..2265
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGATSLSV
                     SVSELCWKQHSKLK"
     primer_bind     1932..1952
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1934..1952
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1999..2018)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2000..2018)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2028..2045)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2028..2044)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2037..2059)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2341..2359)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2427..2531
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2753..3544
                     /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      3715..4303
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4204..4223
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4457..4474
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ggtctcattc ggcccattta agttgaaaac aatcttcaaa agtcccacat cgcttagata
       61 agaaaacgaa gctgagttta tatacagcta gagtcgaagt agtgattgaa aagcaggtgt
      121 ctagtggccg cattaggcac cccaggcttt acactttatg cttccggctc gtataatgtg
      181 tggattttga gttaggatcc gtcgagattt tcaggagcta aggaagctaa aatggagaaa
      241 aaaatcactg gatataccac cgttgatata tcccaatggc atcgtaaaga acattttgag
      301 gcatttcagt cagtcgctca atgtacctat aaccagaccg ttcagctgga tattacggcc
      361 tttttaaaga ccgtaaagaa aaataagcac aagttttatc cggcctttat tcacattctt
      421 gcccgcctga tgaatgctca tccggaattc cgtatggcaa tgaaagacgg tgagctggtg
      481 atatgggata gtgttcaccc ttgttacacc gttttccatg agcaaactga aacgttttca
      541 tcgctctgga gtgaatacca cgacgatttc cggcagtttc tacacatata ttcgcaagat
      601 gtggcgtgtt acggtgaaaa cctggcctat ttccctaaag ggtttattga gaatatgttt
      661 ttcgtttcag ccaatccctg ggtgagtttc accagttttg atttaaacgt ggccaatatg
      721 gacaacttct tcgcccccgt tttcaccatg ggcaaatatt atacgcaagg cgacaaggtg
      781 ctgatgccgc tggcgattca ggttcatcat gccgtttgtg atggcttcca tgtcggcaga
      841 atgcttaatg aattacaaca gtactgcgat gagtggcagg cggggcgtaa tctagaggat
      901 ccggcttact aaaagccaga taacagtatg cgtatttgcg cgctgatttt tgcggtataa
      961 gaatatatac tgatatgtat acccgaagta tgtcaaaagg aggtatgcta tgaagcagcg
     1021 tattacagtg acagttgaca gcgacagcta tcagttgctc aaggcatata tgatgtcaat
     1081 atctccggtc tggtaagcac aaccatgcag aatgaagccc gtcgtctgcg tgccgaacgc
     1141 tggaaagcgg aaaatcagga agggatggct gaggtcgccc ggtttattga aatgaacggc
     1201 tcttttgctg acgagaacag gggctggtga aatgcagttt aaggtttaca cctataaaag
     1261 agagagccgt tatcgtctgt ttgtggatgt acagagtgat attattgaca cgcccgggcg
     1321 acggatggtg atccccctgg ccagtgcacg tctgctgtca gataaagtct cccgtgaact
     1381 ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg atatggccag
     1441 tgtgccggtt tccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat
     1501 caaaaacgcc attaacctga tgttctgggg aatataacac tagacacctg caaaagtttc
     1561 agagctatgc tggaaacagc atagcaagtt gaaataaggc tagtccgtta tcaacttgaa
     1621 aaagtggcac cgagtcggtg cttttttttg gatccgccca tttaagttga aaacaatctt
     1681 caaaagtccc acatcgctta gataagaaaa cgaagctgag tttatataca gctagagtcg
     1741 aagtagtgat tgagagacgg ataaactagt ggaaagcggg cagtgagcgc aacgcaatta
     1801 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
     1861 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
     1921 acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggaggac ctcgaggggg
     1981 ggcccggtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt
     2041 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     2101 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     2161 ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgccactag tttatccgtc
     2221 tcagtttcag agctatgctg gaaacagcat agcaagttga aataaggcta gtccgttatc
     2281 aacttgaaaa agtggcaccg agtcggtgct ttttttttgg cagagaccgt ggacgaaagg
     2341 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     2401 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     2461 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     2521 aaaggaagag tatgcgctca cgcaactggt ccagaacctt gaccgaacgc agcggtggta
     2581 acggcgcagt ggcggttttc atggcttgtt atgactgttt ttttttgggg tacagtctat
     2641 gcctcgggca tccaagcagc aagcgcgtta cgccgtgggt cgatgtttga tgttatggag
     2701 cagcaacgat gttacgcagc agggcagtcg ccctaaaaca aagttaaaca tcatgaggga
     2761 agcggtgatc gccgaagtat cgactcaact atcagaggta gttggcgtca tcgagcgcca
     2821 tctcgaaccg acgttgctgg ccgtacattt gtacggctcc gcagtggatg gcggcctgaa
     2881 gccacacagc gatattgatt tgctggttac ggtgaccgta aggcttgatg aaacaacgcg
     2941 gcgagctttg atcaacgacc ttttggaaac ttcggcttcc cctggagaga gcgagattct
     3001 ccgcgctgta gaagtcacca ttgttgtgca cgacgacatc attccgtggc gttatccagc
     3061 taagcgcgaa ctgcaatttg gagaatggca gcgcaatgac attcttgcag gtatcttcga
     3121 gccagccacg atcgacattg atctggctat cttgctgaca aaagcaagag aacatagcgt
     3181 tgccttggta ggtccagcgg cggaggaact ctttgatccg gttcctgaac aggatctatt
     3241 tgaggcgcta aatgaaacct taacgctatg gaactcgccg cccgactggg ctggcgatga
     3301 gcgaaatgta gtgcttacgt tgtcccgcat ttggtacagc gcagtaaccg gcaaaatcgc
     3361 gccgaaggat gtcgctgccg actgggcaat ggagcgcctg ccggcccagt atcagcccgt
     3421 catacttgaa gctagacagg cttatcttgg acaagaagaa gatcgcttgg cctcgcgcgc
     3481 agatcagttg gaagaatttg tccattacgt aaaaggcgag atcaccaagg tagtcggcaa
     3541 ataactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     3601 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     3661 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     3721 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     3781 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     3841 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     3901 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     3961 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     4021 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     4081 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     4141 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     4201 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     4261 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     4321 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     4381 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     4441 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagc
//
