LOCUS       sequence-438190-        5313 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level P Golden gate accepter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5313)
  AUTHORS   Lawrenson T, Youles M, Chhetry M, Clarke M, Harwood W, Hundleby P
  TITLE     Efficient Targeted Mutagenesis in Brassica Crops Using CRISPR/Cas 
            Systems.
  JOURNAL   Methods Mol Biol. 2023;2653:253-271. doi: 
            10.1007/978-1-0716-3131-7_16.
  PUBMED    36995631
REFERENCE   2  (bases 1 to 5313)
  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: "Methods Mol
            Biol."; date: "2023"; volume: "2653:253-271. doi"; pages: " 
            10.1007/978-1-0716-3131-7_16"
COMMENT     Sequence Label: pICSL4723-P1
FEATURES             Location/Qualifiers
     source          1..5313
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    123..147
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     protein_bind    292..313
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(328..333,334..351,352..358)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    366..382
                     /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     371..393
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     390..406
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     390..406
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             402..725
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    419..475
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(476..493)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(476..492)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(485..507)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(701..720)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     misc_feature    856..880
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             1189..1818
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             2247..3320
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="MSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
                     QAAADRIRKESRQPPAAGASSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
                     KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
                     GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
                     ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
                     LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
                     LVMRYRNLIEGEASAGS"
     rep_origin      3386..3580
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     promoter        3642..3746
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3747..4541
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSLREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     rep_origin      4712..5300
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5201..5220
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cggccttttt acggttcctg ctcggatctg ttggaccgga cagtagtcat ggttgatggg
       61 ctgcctgtat cgagtggtga ttttgtgccg agctgccggt cggggagctg ttggctggct
      121 ggtggcagga tatattgtgg tgtaaacaaa ttgacgctta gacaacttaa taacacattg
      181 cggacgtttt taatgtactg gggttgaaca ctctgtggaa gacaatgcct gagaccgcag
      241 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
      301 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
      361 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
      421 gcttgcatgc ctgcaggtcg actctagagg atccccgggt accgagctcg aattcactgg
      481 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
      541 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
      601 cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc
      661 atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg
      721 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
      781 tgctcccggc atccgcttac agacaagctg tgaggtctca gggacacgaa gtgatccgtt
      841 taaactatca gtgtttgaca ggatatattg gcgggtaaac ctaagagaaa agagcgttta
      901 ttagaataat cggatattta aaagggcgtg aaaaggttta tccgttcgtc catttgtatg
      961 tgcatgccaa ccacagggtt cccctcggga gtcagccgtg cggctgcatg aaatcctggc
     1021 cggtttgtct gatgccaagc tggcggcctg gccggccagc ttggccgctg aagaaaccga
     1081 gcgccgccgt ctaaaaaggt gatgtgtatt tgagtaaaac agcttgcgtc atgcggtcgc
     1141 tgcgtatatg atgcgatgag taaataaaca aatacgcaag gggaacgcat gaaggttatc
     1201 gctgtactta accagaaagg cgggtcaggc aagacgacca tcgcaaccca tctagcccgc
     1261 gccctgcaac tcgccggggc cgatgttctg ttagtcgatt ccgatcccca gggcagtgcc
     1321 cgcgattggg cggccgtgcg ggaagatcaa ccgctaaccg ttgtcggcat cgaccgcccg
     1381 acgattgacc gcgacgtgaa ggccatcggc cggcgcgact tcgtagtgat cgacggagcg
     1441 ccccaggcgg cggacttggc tgtgtccgcg atcaaggcag ccgacttcgt gctgattccg
     1501 gtgcagccaa gcccttacga catatgggcc accgccgacc tggtggagct ggttaagcag
     1561 cgcattgagg tcacggatgg aaggctacaa gcggcctttg tcgtgtcgcg ggcgatcaaa
     1621 ggcacgcgca tcggcggtga ggttgccgag gcgctggccg ggtacgagct gcccattctt
     1681 gagtcccgta tcacgcagcg cgtgagctac ccaggcactg ccgccgccgg cacaaccgtt
     1741 cttgaatcag aacccgaggg cgacgctgcc cgcgaggtcc aggcgctggc cgctgaaatt
     1801 aaatcaaaac tcatttgagt taatgaggta aagagaaaat gagcaaaagc acaaacacgc
     1861 taagtgccgg ccgtccgagc gcacgcagca gcaaggctgc aacgttggcc agcctggcag
     1921 acacgccagc catgaagcgg gtcaactttc agttgccggc ggaggatcac accaagctga
     1981 agatgtacgc ggtacgccaa ggcaagacca ttaccgagct gctatctgaa tacatcgcgc
     2041 agctaccaga gtaaatgagc aaatgaataa atgagtagat gaattttagc ggctaaagga
     2101 ggcggcatgg aaaatcaaga acaaccaggc accgacgccg tggaatgccc catgtgtgga
     2161 ggaacgggcg gttggccagg cgtaagcggc tgggttgtct gccggccctg caatggcact
     2221 ggaaccccca agcccgagga atcggcgtga gcggtcgcaa accatccggc ccggtacaaa
     2281 tcggcgcggc gctgggtgat gacctggtgg agaagttgaa ggccgcgcag gccgcccagc
     2341 ggcaacgcat cgaggcagaa gcacgccccg gtgaatcgtg gcaagcggcc gctgatcgaa
     2401 tccgcaaaga atcccggcaa ccgccggcag ccggtgcgtc gtcgattagg aagccgccca
     2461 agggcgacga gcaaccagat tttttcgttc cgatgctcta tgacgtgggc acccgcgata
     2521 gtcgcagcat catggacgtg gccgttttcc gtctgtcgaa gcgtgaccga cgagctggcg
     2581 aggtgatccg ctacgagctt ccagacgggc acgtagaggt ttccgcaggg ccggccggca
     2641 tggccagtgt gtgggattac gacctggtac tgatggcggt ttcccatcta accgaatcca
     2701 tgaaccgata ccgggaaggg aagggagaca agcccggccg cgtgttccgt ccacacgttg
     2761 cggacgtact caagttctgc cggcgagccg atggcggaaa gcagaaagac gacctggtag
     2821 aaacctgcat tcggttaaac accacgcacg ttgccatgca gcgtacgaag aaggccaaga
     2881 acggccgcct ggtgacggta tccgagggtg aagccttgat tagccgctac aagatcgtaa
     2941 agagcgaaac cgggcggccg gagtacatcg agatcgagct agctgattgg atgtaccgcg
     3001 agatcacaga aggcaagaac ccggacgtgc tgacggttca ccccgattac tttttgatcg
     3061 atcccggcat cggccgtttt ctctaccgcc tggcacgccg cgccgcaggc aaggcagaag
     3121 ccagatggtt gttcaagacg atctacgaac gcagtggcag cgccggagag ttcaagaagt
     3181 tctgtttcac cgtgcgcaag ctgatcgggt caaatgacct gccggagtac gatttgaagg
     3241 aggaggcggg gcaggctggc ccgatcctag tcatgcgcta ccgcaacctg atcgagggcg
     3301 aagcatccgc cggttcctaa tgtacggagc agatgctagg gcaaattgcc ctagcagggg
     3361 aaaaaggtcg aaaaagcttc tttcctgtgg atagcacgta cattgggaac ccaaagccgt
     3421 acattgggaa ccggaacccg tacattggga acccaaagcc gtacattggg aaccggtcac
     3481 acatgtaagt gactgatata aaagagaaaa aaggcgattt ttccgcctaa aactctttaa
     3541 aacttattaa aactcttaaa acccgcctgg cctgtgcata actgtctggc cagcgcacag
     3601 ccgaacagct gcaaaaagcg cccacatcaa ggctccgagt gcgcggaacc cctatttgtt
     3661 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     3721 ttcaataata ttgaaaaagg aagagtatgg ctaaaatgag aatatcaccg gaattgaaaa
     3781 aactgatcga aaaataccgc tgcgtaaaag atacggaagg aatgtctcct gctaaggtat
     3841 ataagctggt gggagaaaat gaaaacctat atttaaaaat gacggacagc cggtataaag
     3901 ggaccaccta tgatgtggaa cgggaaaagg acatgatgct atggctggaa ggaaagctgc
     3961 ctgttccaaa ggtcctgcac tttgaacggc atgatggctg gagcaatctg ctcatgagtg
     4021 aggccgatgg cgtcctttgc tcggaagagt atgaagatga acaaagccct gaaaagatta
     4081 tcgagctgta tgcggagtgc atcaggctct ttcactccat cgacatatcg gattgtccct
     4141 atacgaatag cttagacagc cgcttagccg aattggatta cttactgaat aacgatctgg
     4201 ccgatgtgga ttgcgaaaac tgggaagagg acactccatt taaagatccg cgcgagctgt
     4261 atgatttttt aaagacggaa aagcccgaag aggaacttgt cttttcccac ggcgacctgg
     4321 gagacagcaa catctttgtg aaagatggca aagtaagtgg ctttattgat cttgggagaa
     4381 gcggcagggc ggacaagtgg tatgacattg ccttctgcgt ccggtcgctc agggaggata
     4441 tcggggaaga acagtatgtc gagctatttt ttgacttact ggggatcaag cctgattggg
     4501 agaaaataaa atattatatt ttactggatg aattgtttta gctgtcagac caagtttact
     4561 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     4621 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     4681 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     4741 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     4801 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     4861 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     4921 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     4981 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     5041 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     5101 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     5161 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     5221 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     5281 gggggcggag cctatggaaa aacgccagca acg
//
