LOCUS       sequence-209247-        3612 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  more efficient version of pFH6; Subcloning of any sgRNA via BbsI 
            site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3612)
  AUTHORS   Hahn F, Eisenhut M, Mantegazza O, Weber APM
  TITLE     Generation of Targeted Knockout Mutants in Arabidopsis thaliana 
            Using CRISPR/Cas9.
  JOURNAL   Bio Protoc. 2017 Jul 5;7(13):e2384. doi: 10.21769/BioProtoc.2384. 
            eCollection 2017 Jul 5.
  PUBMED    34541122
REFERENCE   2  (bases 1 to 3612)
  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: "Bio 
            Protoc."; date: "2017-07-5"; volume: "7(13):e2384. doi"; pages: " 
            10.21769/BioProtoc.2384. eCollection 2017 Jul 5"
COMMENT     Sequence Label: pFH6_new
FEATURES             Location/Qualifiers
     source          1..3612
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(26..817,818..886))
                     /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     649..668
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(887..991)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1069..1524)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1206..1227)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1418..1437
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1650..1672
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1664..1681
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1665..1681
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1688..1707
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1688..1706
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1737..1746
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1741..1750
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_RNA        2237..2312
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(2441..2459)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2442..2459)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2477..2493)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2477..2493)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2490..2512)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2501..2517
                     /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(2525..2531,2532..2549,2550..2555))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2570..2591
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2708..2725)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2879..3467)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2959..2978)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
       61 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
      121 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
      181 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
      241 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
      301 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
      361 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
      421 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
      481 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
      541 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
      601 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
      661 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
      721 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
      781 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
      841 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
      901 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
      961 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgatg
     1021 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggaa attgtaagcg
     1081 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     1141 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     1201 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     1261 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     1321 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     1381 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     1441 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     1501 ttaatgcgcc gctacagggc gcgtccattc gccattcagg ctgcgcaact gttgggaagg
     1561 gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag
     1621 gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag
     1681 tgaattgtaa tacgactcac tatagggcga attgggcccg acgtcgcatg ctcccggccg
     1741 ccatggcggc cgcgggaatt cgattcacga agcttcgttg aacaacggaa actcgacttg
     1801 ccttccgcac aatacatcat ttcttcttag ctttttttct tcttcttcgt tcatacagtt
     1861 tttttttgtt tatcagctta cattttcttg aaccgtagct ttcgttttct tctttttaac
     1921 tttccattcg gagtttttgt atcttgtttc atagtttgtc ccaggattag aatgattagg
     1981 catcgaacct tcaagaattt gattgaataa aacatcttca ttcttaagat atgaagataa
     2041 tcttcaaaag gcccctggga atctgaaaga agagaagcag gcccatttat atgggaaaga
     2101 acaatagtat ttcttatata ggcccattta agttgaaaac aatcttcaaa agtcccacat
     2161 cgcttagata agaaaacgaa gctgagttta tatacagcta gagtcgaagt agtgattggg
     2221 gtcttcgaga agacctgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt
     2281 atcaacttga aaaagtggca ccgagtcggt gcaaattttt ttaaactagt ggtgatcttg
     2341 ctgaaaaact cgagccatcc ggaagatctt gaattcgcgg ccgcctgcag gtcgaccata
     2401 tgggagagct cccaacgcgt tggatgcata gcttgagtat tctatagtgt cacctaaata
     2461 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     2521 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     2581 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     2641 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     2701 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     2761 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     2821 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     2881 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     2941 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     3001 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     3061 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     3121 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     3181 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     3241 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     3301 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     3361 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     3421 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     3481 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     3541 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     3601 caatctaaag ta
//
