LOCUS       sequence-331273-        3568 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GoldenGate (MoClo) Level 1 Position 4 TaU6 guide acceptor plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3568)
  AUTHORS   Smedley MA, Hayta S, Clarke M, Harwood WA
  TITLE     CRISPR-Cas9 Based Genome Editing in Wheat.
  JOURNAL   Curr Protoc. 2021 Mar;1(3):e65. doi: 10.1002/cpz1.65.
  PUBMED    33687760
REFERENCE   2  (bases 1 to 3568)
  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: "Curr 
            Protoc."; date: "2021-03"; volume: "1(3):e65. doi"; pages: " 
            10.1002/cpz1.65"
COMMENT     Sequence Label: Level 1 P4 TaU6 guide acceptor
FEATURES             Location/Qualifiers
     source          1..3568
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    575..596
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(611..616,617..634,635..641)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    649..665
                     /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     673..689
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     673..689
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             685..1008
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    702..758
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(759..776)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(759..775)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(768..790)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     misc_RNA        1105..1180
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(1192..1216)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    1287..1303
                     /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(1311..1317,1318..1335,1336..1341))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1356..1377
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1494..1511)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1665..2253)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1745..1764)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2424..3215,3216..3284))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3047..3066
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3285..3389)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3457..3475
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3513..3535)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta
       61 actatgcggc atcagagcag attgtactga gagtttggca attggtcgac ctcgagggcg
      121 cgcccgtaca ctctgtggaa gacaattacg accaagcccg ttattctgac agttctggtg
      181 ctcaacacat ttatatttat caaggagcac attgttactc actgctagga gggaatcgaa
      241 ctaggaatat tgatcagagg aactacgaga gagctgaaga taactgccct ctagctctca
      301 ctgatctggg tcgcatagtg agatgcagcc cacgtgagtt cagcaacggt ctagcgctgg
      361 gcttttaggc ccgcatgatc gggcttttgt cgggtggtcg acgtgttcac gattggggag
      421 agcaacgcag cagttcctct tagtttagtc ccacctcgcc tgtccagcag agttctgacc
      481 ggtttataaa ctcgcttgct gcatcagact tgtgagaccg cagctggcac gacaggtttc
      541 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
      601 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
      661 aacaatttca cacaggaaac agctatgacc atgattacgc caagcttgca tgcctgcagg
      721 tcgactctag aggatccccg ggtaccgagc tcgaattcac tggccgtcgt tttacaacgt
      781 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
      841 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
      901 ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
      961 caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     1021 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     1081 tacagacaag ctgtgacggt ctcagtttta gagctagaaa tagcaagtta aaataaggct
     1141 agtccgttat caacttgaaa aagtggcacc gagtcggtgc tttttttcta gacccagctt
     1201 tcttgtacaa agttggcatt acgctttacc agaaagtctt ccacgaagtg gtggcagctc
     1261 tagagctagc gaattctttg gtgaaattgt tatccgctca caattccaca caacatacga
     1321 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     1381 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     1441 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     1501 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     1561 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     1621 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     1681 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     1741 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     1801 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     1861 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     1921 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     1981 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     2041 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     2101 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     2161 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     2221 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     2281 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     2341 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     2401 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     2461 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     2521 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgcgaacc acgctcaccg
     2581 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     2641 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     2701 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     2761 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     2821 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     2881 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     2941 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     3001 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     3061 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     3121 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     3181 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     3241 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     3301 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     3361 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     3421 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     3481 cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     3541 gtcacagctt gtctgtaagc ggatgccg
//
