LOCUS       sequence-461762-        3036 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  M13 phagemid that expresses sgRNA-2 from a strong constitutive 
            promoter. (This plasmid is a gift of the Jaramillo Lab, where it is 
            called PAJ171.).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3036)
  AUTHORS   Kushwaha M, Singh V, Jaramillo A
  TITLE     Development of a genetically-encoded in vivo computing platform to 
            play the game of Tic-Tac-Toe
  JOURNAL   Zenodo (2017) Conference: SB7.0: The Seventh International Meeting 
            on Synthetic Biology.
REFERENCE   2  (bases 1 to 3036)
  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: "Zenodo 
            (2017) Conference: SB7.0: The Seventh International Meeting on 
            Synthetic Biology."
COMMENT     Sequence Label: pSEVA26_sgRNA-2_M13ps
FEATURES             Location/Qualifiers
     source          1..3036
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     9..31
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     promoter        62..96
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     misc_RNA        117..192
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      257..351
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(472..491)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             483..1298
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     oriT            1465..1573
                     /note="incP origin of transfer"
     rep_origin      1620..2165
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     2066..2085
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2282..2299
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2332..2841)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2527..2548)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2735..2754
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(2944..3030)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ttaattaaag cggataacaa tttcacacag gaggccgcct aggccgcggc cgcgcgaatt
       61 cttgacagct agctcagtcc taggtataat gctagcttta cacccttgag ctgctagttt
      121 tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca
      181 ccgagtcggt gcttttctgc aggcatgcaa gcttgcggcc gcgtcgtgac tgggaaaacc
      241 ctggcgacta gtcttggact cctgttgata gatccagtaa tgacctcaga actccatctg
      301 gatttgttca gaacgctcgg ttgccgccgg gcgtttttta ttggtgagaa tccaggggtc
      361 cccaataatt acgatttaaa tttgtgtctc aaaatctctg atgttacatt gcacaagata
      421 aaaatatatc atcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg
      481 ttatgagcca tattcagcgt gaaacgagct gtagccgtcc gcgtctgaac agcaacatgg
      541 atgcggatct gtatggctat aaatgggcgc gtgataacgt gggtcagagc ggcgcgacca
      601 tttatcgtct gtatggcaaa ccggatgcgc cggaactgtt tctgaaacat ggcaaaggca
      661 gcgtggcgaa cgatgtgacc gatgaaatgg tgcgtctgaa ctggctgacc gaatttatgc
      721 cgctgccgac cattaaacat tttattcgca ccccggatga tgcgtggctg ctgaccaccg
      781 cgattccggg caaaaccgcg tttcaggtgc tggaagaata tccggatagc ggcgaaaaca
      841 ttgtggatgc gctggccgtg tttctgcgtc gtctgcatag cattccggtg tgcaactgcc
      901 cgtttaacag cgatcgtgtg tttcgtctgg cccaggcgca gagccgtatg aacaacggcc
      961 tggtggatgc gagcgatttt gatgatgaac gtaacggctg gccggtggaa caggtgtgga
     1021 aagaaatgca taaactgctg ccgtttagcc cggatagcgt ggtgacccac ggcgatttta
     1081 gcctggataa cctgattttc gatgaaggca aactgattgg ctgcattgat gtgggccgtg
     1141 tgggcattgc ggatcgttat caggatctgg ccattctgtg gaactgcctg ggcgaattta
     1201 gcccgagcct gcaaaaacgt ctgtttcaga aatatggcat tgataatccg gatatgaaca
     1261 aactgcaatt tcatctgatg ctggatgaat ttttctaata attaattgga ccgcggtccg
     1321 cgcgttgtcc ttttccgctg cataaccctg cttcggggtc attatagcga ttttttcggt
     1381 atatccatcc tttttcgcac gatatacagg attttgccaa agggttcgtg tagactttcc
     1441 ttggtgtatc caacggcgtc agccgggcag gataggtgaa gtaggcccac ccgcgagcgg
     1501 gtgttccttc ttcactgtcc cttattcgca cctggcggtg ctcaacggga atcctgctct
     1561 gcgaggctgg ccgtaggccg gccctagaaa tattttatct gattaataag atgatcttct
     1621 tgagatcgtt ttggtctgcg cgtaatctct tgctctgaaa acgaaaaaac cgccttgcag
     1681 ggcggttttt cgaaggttct ctgagctacc aactctttga accgaggtaa ctggcttgga
     1741 ggagcgcagt caccaaaact tgtcctttca gtttagcctt aaccggcgca tgacttcaag
     1801 actaactcct ctaaatcaat taccagtggc tgctgccagt ggtgcttttg catgtctttc
     1861 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcggact gaacgggggg
     1921 ttcgtgcata cagtccagct tggagcgaac tgcctacccg gaactgagtg tcaggcgtgg
     1981 aatgagacaa acgcggccat aacagcggaa tgacaccggt aaaccgaaag gcaggaacag
     2041 gagagcgcac gagggagccg ccagggggaa acgcctggta tctttatagt cctgtcgggt
     2101 ttcgccacca ctgatttgag cgtcagattt cgtgatgctt gtcagggggg cggagcctat
     2161 ggaaaaacgg ctttgccgcg gccctctcac ttccctgtta agtatcttcc tggcatcttc
     2221 caggaaatct ccgccccgtt cgtaagccat ttccgctcgc cgcagtcgaa cgaccgagcg
     2281 tagcgagtca gtgagcgagg aagcggaata tatccggcgc gagattgatt taccccggtt
     2341 gataatcaga aaagccccaa aaacaggaag attgtataag caaatattta aattgtaaac
     2401 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     2461 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agcccgagat agggttgagt
     2521 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     2581 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccaa atcaagtttt
     2641 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     2701 gcttgacggg gaaagcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     2761 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     2821 atgcgccgct acagggcgcg taaaaggatc taggtgaaga tcctttttga taatctcatg
     2881 accaaaatcc cttaacgtga gttttcgttc cactgaggcg cgcccagctg tctagggcgg
     2941 cggatttgtc ctactcagga gagcgttcac cgacaaacaa cagataaaac gaaaggccca
     3001 gtctttcgac tgagcctttc gttttatttg atgcct
//
