pJOE8999

LOCUS       pJOE8999                7794 bp    DNA     circular SYN 19-JUN-2026
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7794)
  AUTHORS   DotmaticsRD
  TITLE     Direct Submission
  JOURNAL   Exported Jun 19, 2026 from SnapGene 8.1.0
            https://www.biofeng.com
COMMENT     Sequence Label: pJOE8999
FEATURES             Location/Qualifiers
     source          1..7794
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(37..4143)
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /note="color: #993366"                     
     rep_origin      4414..5002
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     primer_bind     7352..7368
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: RIGHT"
     primer_bind     complement(7384..7400)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: LEFT"
     protein_bind    7408..7424
                     /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)."
                     /note="color: #31849b"
     promoter        complement(join(7432..7438,7439..7456,7457..7462))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
                     /note="This reverse directional feature has 3 segments:
                        1: 7432 .. 7438 / #ffffff / -10
                        2: 7439 .. 7456 / #ffffff
                        3: 7457 .. 7462 / #ffffff / -35"
     protein_bind    7477..7498
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
                     /note="color: #31849b"
     misc_RNA        7572..7647
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
                     /note="color: #00ccff; direction: RIGHT"
     terminator      7669..7703
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
                     /note="color: #ffffff; direction: RIGHT"
     promoter        7740..7758
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /note="color: #ffffff; direction: RIGHT"
ORIGIN
        1 tctagattaa gaaataatct tcatctaaaa tatacttcag tcacctccta gctgactcaa
       61 atcaatgcgt gtttcataaa gaccagtgat ggattgatgg ataagagtgg catctaaaac
      121 ttcttttgta gacgtatatc gtttacgatc aattgttgta tcaaaatatt taaaagcagc
      181 gggagctcca agattcgtca acgtaaataa atgaataata ttttctgctt gttcacgtat
      241 tggtttgtct ctatgtttgt tatatgcact aagaacttta tctaaattgg catctgctaa
      301 aataacacgc ttagaaaatt cactgatttg ctcaataatc tcatctaaat aatgcttatg
      361 ctgctccaca aacaattgtt tttgttcgtt atcttctgga ctacccttca acttttcata
      421 atgactagct aaatataaaa aattcacata tttgcttggc agagccagct catttccttt
      481 ttgtaattct ccggcactag ccagcatccg tttacgaccg ttttctaact caaaaagact
      541 atatttaggt agtttaatga ttaagtcttt tttaacttcc ttatatcctt tagcttctaa
      601 aaagtcaatc ggattttttt caaaggaact tctttccata attgtgatcc ctagtaactc
      661 tttaacggat tttaacttct tcgatttccc tttttccacc ttagcaacca ctaggactga
      721 ataagctacc gttggactat caaaaccacc atattttttt ggatcccagt cttttttacg
      781 agcaataagc ttgtccgaat ttctttttgg taaaattgac tccttggaga atccgcctgt
      841 ctgtacttct gttttcttga caatattgac ttggggcatg gacaatactt tgcgcactgt
      901 ggcaaaatct cgccctttat cccagacaat ttctccagtt tccccattag tttcgattag
      961 agggcgtttg cgaatctctc catttgcaag tgtaatttct gttttgaaga agttcatgat
     1021 attagagtaa aagaaatatt ttgcggttgc tttgcctatt tcttgctcag acttagcaat
     1081 cattttacga acatcataaa ctttataatc accatagaca aactccgatt caagttttgg
     1141 atatttctta atcaaagcag ttccaacgac ggcatttaga tacgcatcat gggcatgatg
     1201 gtaattgtta atctcacgta ctttatagaa ttggaaatct tttcggaagt cagaaactaa
     1261 tttagatttt aaggtaatca ctttaacctc tcgaataagt ttatcatttt catcgtattt
     1321 agtattcatg cgactatcca aaatttgtgc cacatgctta gtgatttggc gagtttcaac
     1381 caattggcgt ttgataaaac cagctttatc aagttcactc aaacctccac gttcagcttt
     1441 cgttaaatta tcaaacttac gttgagtgat taacttggcg tttagaagtt gtctccaata
     1501 gtttttcatc tttttgacta cttcttcact tggaacgtta tccgatttac cacgattttt
     1561 atcagaacgc gttaagacct tattgtctat tgaatcgtct ttaaggaaac tttgtggaac
     1621 aatgtgatcg acatcataat cacttaaacg attaatatct aattcttggt ccacatacat
     1681 gtctcttcca ttttggagat aatagagata gagcttttca ttttgcaatt gagtattttc
     1741 aacaggatgc tctttaagaa tctgacttcc taattctttg ataccttctt cgattcgttt
     1801 catacgctct cgcgaatttt tctggccctt ttgagttgtc tgattttcac gtgccatttc
     1861 aataacgata ttttctggct tatgccgccc cattactttg accaattcat caacaacttt
     1921 tacagtctgt aaaatacctt ttttaatagc agggctacca gctaaatttg caatatgttc
     1981 atgtaaacta tcgccttgtc cagacacttg tgctttttga atgtcttctt taaatgtcaa
     2041 actatcatca tggatcagct gcataaaatt gcgattggca aaaccatctg atttcaaaaa
     2101 atctaatatt gttttgccag attgcttatc cctaatacca ttaatcaatt ttcgagacaa
     2161 acgtccccaa ccagtataac ggcgacgttt aagctgtttc atcaccttat catcaaagag
     2221 gtgagcatat gttttaagtc tttcctcaat catctcccta tcttcaaata aggtcaatgt
     2281 taaaacaata tcctctaaga tatcttcatt ttcttcatta tccaaaaaat ctttatcttt
     2341 aataattttt agcaaatcat ggtaggtacc taatgaagca ttaaatctat cttcaactcc
     2401 tgaaatttca acactatcaa aacattctat ttttttgaaa taatcttctt ttaattgctt
     2461 aacggttact tttcgatttg ttttgaagag taaatcaaca atggctttct tctgttcacc
     2521 tgaaagaaat gctggttttc gcattccttc agtaacatat ttgacctttg tcaattcgtt
     2581 ataaaccgta aaatactcat aaagcaaact atgttttggt agtacttttt catttggaag
     2641 atttttatca aagtttgtca tgcgttcaat aaatgattga gctgaagcac ctttatcgac
     2701 aacttcttca aaattccatg gggtaattgt ttcttcagac ttccgagtca tccatgcaaa
     2761 acgactattg ccacgcgcca atggaccaac ataataagga attcgaaaag tcaagatttt
     2821 ttcaatcttc tcacgattgt cttttaaaaa tggataaaag tcttcttgtc ttctcaaaat
     2881 agcatgcagc tcacccaagt gaatttgatg gggaatagag ccgttgtcaa aggtccgttg
     2941 cttgcgcagc aaatcttcac gatttagttt caccaataat tcctcagtac catccatttt
     3001 ttctaaaatt ggtttgataa atttataaaa ttcttcttgg ctagctcccc catcaatata
     3061 acctgcatat ccgttttttg attgatcaaa aaagatttct ttatactttt ctggaagttg
     3121 ttgtcgaact aaagctttta aaagagtcaa gtcttgatga tgttcatcgt agcgtttaat
     3181 cattgaagct gataggggag ccttagttat ttcagtattt actcttagga tatctgaaag
     3241 taaaatagca tctgataaat tcttagctgc caaaaacaaa tcagcatatt gatctccaat
     3301 ttgcgccaat aaattatcta aatcatcatc gtaagtatct tttgaaagct gtaatttagc
     3361 atcttctgcc aaatcaaaat ttgatttaaa attaggggtc aaacccaatg acaaagcaat
     3421 gagattccca aataagccat ttttcttctc accggggagc tgagcaatga gattttctaa
     3481 tcgtcttgat ttactcaatc gtgcagaaag aatcgcttta gcatctactc cacttgcgtt
     3541 aatagggttt tcttcaaata attgattgta ggtttgtacc aactggataa atagtttgtc
     3601 cacatcacta ttatcaggat ttaaatctcc ctcaatcaaa aaatgaccac gaaacttaat
     3661 catatgcgct aaggccaaat agattaagcg caaatccgct ttatcagtag aatctaccaa
     3721 tttttttcgc agatgataga tagttggata tttctcatga taagcaactt catctactat
     3781 atttccaaaa ataggatgac gttcatgctt cttgtcttct tccaccaaaa aagactcttc
     3841 aagtcgatga aagaaactat catctacttt cgccatctca tttgaaaaaa tctcctgtag
     3901 ataacaaata cgattcttcc gacgtgtata ccttctacga gctgtccgtt tgagacgagt
     3961 cgcttccgct gtctctccac tgtcaaataa aagagcccct ataagatttt ttttgatact
     4021 gtggcggtct gtatttccca gaaccttgaa ctttttagac ggaaccttat aatcatcagt
     4081 gatcaccgcc catccgacgc tatttgtgcc gatatctaag cctattgagt atttcttatc
     4141 catttttgcc tcctaactta agaataagat cttgtctcaa ctgtataccg aaatcagctc
     4201 attaaaatcg ctttttttac cataggttcc ggtaataaag gcatttttcc ctataacaaa
     4261 aaaagcaagg aataatccct gcttttaata atccaaatga gataaaaatg tcatgacatt
     4321 ggtgtacaga agaatggcca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     4381 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     4441 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     4501 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     4561 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     4621 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     4681 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     4741 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     4801 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     4861 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     4921 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     4981 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     5041 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     5101 tattaccgcc tttgagtgag ctgaaattat gaggggatct ctcagagctc gaggtcatcg
     5161 ttcaaaatgg tatgcgtttt gacacatcca ctatatatcc gtgtcgttct gtccactcct
     5221 gaatcccatt ccagaaattc tctagcgatt ccagaagttt ctcagagtcg gaaagttgac
     5281 cagacattac gaactggcac agatggtcat aacctgaagg aagatctgat tgcttaactg
     5341 cttcagttaa gaccgaagcg ctcgtcgtat aacagatgcg atgatgcaga ccaatcaaca
     5401 tggcacctgc cattgctacc tgcacagtca aggatggtag aaatgttgtc ggtccttgca
     5461 cacgaatatt acgccatttg cctgcatatt caaacagctc ttctacgata agggcacaaa
     5521 tcgcatcgtg gaacgtttgg gcttctaccg atttagcagt ttgatacact ttctctaagt
     5581 atccacctga atcataaatc ggcaaaatag agaaaaattg accatgtgta agcggccaat
     5641 ctgattccac ctgagatgca taatctagta gaatctcttc gctatcaaaa ttcacttcca
     5701 ccttccactc accggttgtc cattcatggc tgaactctgc ttcctctgtt gacatgacac
     5761 acatcatctc aatatccgaa tagggcccat cagtctgacg accaagagag ccataaacac
     5821 caatagcctt aacatcatcc ccatatttat ccaatattcg ttccttaatt tcatgaacaa
     5881 tcttcattct ttcttctcta gtcattatta ttggtccatt cactattctc attccctttt
     5941 cagataattt tagatttgct tttctaaata agaatatttg gagagcaccg ttcttattca
     6001 gctattaaac ccattatatc gggtttttga ggggatttca actgcagaca cctaaattca
     6061 aaatctatcg gtcagattta taccgatttg attttatata ttcttgaata acatacgccg
     6121 agttatcaca taaaagcggg aaccaatcat caaatttaaa cttcattgca taatccatta
     6181 aactcttaaa ttctacgatt ccttgttcat caataaactc aatcatttct ttaattaatt
     6241 tatatctatc tgttgttgtt ttctttaata attcatcaac atctacaccg ccataaacta
     6301 tcatatcttc tttttgatat ttaaatttat taggatcgtc catgtgaagc atatatctca
     6361 caagaccttt cacacttcct gcaatctgcg gaatagtcgc attcaattct tctgttaatt
     6421 atttttatct gttcataaga tttattaccc tcatacatca ctagaatatg ataatgctct
     6481 tttttcatcc taccttctgt atcagtatcc ctatcatgta atggagacac tacaaattga
     6541 atgtgtaact cttttaaata ctctaaccac tcggcttttg ctgattctgg atataaaaca
     6601 aatgtccaat tacgtcctct tgaatttttc ttgttttcag tttcttttat tacattttcg
     6661 ctcatgatat aataacggtg ctaatacatt taacaaaatt tagtcataga taggcagcat
     6721 gccagtgctg tctatctttt tttgtttaaa atgcaccgta ttcctccttt gcatattttt
     6781 ttattagaat accggttgca tctgatttgc taatattata tttttctttg attctattta
     6841 atatctcatt ttcttctgtt gtaagtctta aagtaacagc aacttttttc tcttcttttc
     6901 tatctacaac catcactgta cctcccaaca tctgtttttt tcactttaac ataaaaaaca
     6961 accttttaac attaaaaacc caatatttat ttatttgttt ggacaatgga caatggacac
     7021 ctagggggga ggtcgtagta cccccctatg ttttctcccc taaataaccc caaaaatcta
     7081 agaaaaaaag acctcaaaaa ggtctttaat taacatctca aatttcgcat ttattccaat
     7141 ttcctttttg cgtgtgatgc gaattcttga ccgtgattag agaattgagt aaaatgtacc
     7201 tacgcgagac ctcattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg
     7261 gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg
     7321 gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga attcgtaatc
     7381 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     7441 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     7501 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     7561 aatcggtctc agttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa
     7621 cttgaaaaag tggcaccgag tcggtgcttt ttactccatc tggatttgtt cagaacgctc
     7681 ggttgccgcc gggcgttttt tatctaaagc ttaggcccag tcgaaagact gggccttttt
     7741 aatacgactc actatagggt cgacggccaa cgaggcccgg gccaataagg cctt
//