LOCUS       sequence-260896-        3004 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains three stop codons in every frame to ensure a premature stop
            codon when introduced into the genome via homology-directed repair. 
            Gateway multisite entry clone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3004)
  AUTHORS   Mallett DR, Chang M, Cheng X, Bezanilla M
  TITLE     Efficient and modular CRISPR-Cas9 vector system for Physcomitrella 
            patens.
  JOURNAL   Plant Direct. 2019 Sep 12;3(9):e00168. doi: 10.1002/pld3.168. 
            eCollection 2019 Sep.
  PUBMED    31523744
REFERENCE   2  (bases 1 to 3004)
  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: "Plant 
            Direct."; date: "2019-09-12"; volume: "3(9):e00168. doi"; pages: " 
            10.1002/pld3.168. eCollection 2019 Sep"
COMMENT     Sequence Label: pENTR-R4R3-stop
FEATURES             Location/Qualifiers
     source          1..3004
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(603..726)
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     774..790
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        797..815
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    824..931
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     840..856
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     841..857
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(891..907)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(891..907)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        complement(944..962)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(983..999)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(996..1018)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1007..1023
                     /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(1031..1037,1038..1055,1056..1061))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1067..1190
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(1246..1264)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1269..1285)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1387..1406)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1398..2207
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1465..1484)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2354..2942
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2843..2862
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccct acaggtcact aataccatct aagtagttga
      601 ttcatagtga ctggatatgt tgtgttttac agtattatgt agtctgtttt ttatgcaaaa
      661 tctaatttaa tatattgata tttatatcat tttacgtttc tcgttcaact tttctataca
      721 aagttgtagc tgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac
      781 gacggccagt gaattgtaat acgactcact atagggcgaa ttgggtaccg ggccccccct
      841 cgaggtcgac ggtatcgata agcttgatat cgaattcctg cagcccgggg gatccactag
      901 ttctagagcg gccgccaccg cggtggagct ccagcttttg ttccctttag tgagggttaa
      961 tttcgagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     1021 caattccaca caacatacga gccggaagca taaagtgtaa agcctacaac tttgtataat
     1081 aaagttgaac gagaaacgta aaatgatata aatatcaata tattaaatta gattttgcat
     1141 aaaaaacaga ctacataata ctgtaaaaca caacatatcc agtcactatg aatcaactac
     1201 ttagatggta ttagtgacct gtactgcagg gcggccgcga tatcccctat agtgagtcgt
     1261 attacatggt catagctgtt tcctggcagc tctggcccgt gtctcaaaat ctctgatgtt
     1321 acattgcaca agataaaaat atatcatcat gaacaataaa actgtctgct tacataaaca
     1381 gtaatacaag gggtgttatg agccatattc aacgggaaac gtcgaggccg cgattaaatt
     1441 ccaacatgga tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag
     1501 gtgcgacaat ctatcgcttg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg
     1561 gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg
     1621 aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac
     1681 tcaccactgc gatccccgga aaaacagcat tccaggtatt agaagaatat cctgattcag
     1741 gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt
     1801 gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga
     1861 ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac
     1921 aagtctggaa agaaatgcat aaacttttgc cattctcacc ggattcagtc gtcactcatg
     1981 gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg
     2041 ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg
     2101 gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg
     2161 atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaatca gaattggtta
     2221 attggttgta acactggcag agcattacgc tgacttgacg ggacggcgca agctcatgac
     2281 caaaatccct taacgtgagt tacgcgtcgt tccactgagc gtcagacccc gtagaaaaga
     2341 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     2401 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     2461 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     2521 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     2581 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     2641 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     2701 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     2761 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     2821 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     2881 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     2941 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     3001 tgtt
//
