LOCUS       sequence-461746-        3790 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  M13 phagemid constitutively expressing sgRNA-C0. sgRNA-C0 is a dummy
            20-nt sequence, containing a Golden Gate cloning site (BsaI). (A 
            gift of the Jaramillo Lab, where it is called PAJ156.).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3790)
  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 3790)
  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: pSEVA19_sgRNA-C0_M13ps
FEATURES             Location/Qualifiers
     source          1..3790
                     /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"
     promoter        424..515
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(516..584,585..1376)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     terminator      1380..1419
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     oriT            1566..1674
                     /note="incP origin of transfer"
     rep_origin      1710..2298
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2199..2218
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2452..2469
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2484..2624
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2550..2569)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2710..2732
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2726..2917)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     rep_origin      complement(3086..3595)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3281..3302)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3489..3508
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(3698..3784)
                     /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 gctagcagag accactttcg gtctctgttt
      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 ttagtagccc gcctaatgag cgggcttttt tttaattccc
      421 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
      481 gataaatgct tcaataatat tgaaaaagga agagtatgag cattcagcat tttcgtgtgg
      541 cgctgattcc gttttttgcg gcgttttgcc tgccggtgtt tgcgcatccg gaaaccctgg
      601 tgaaagtgaa agatgcggaa gatcaactgg gtgcgcgcgt gggctatatt gaactggatc
      661 tgaacagcgg caaaattctg gaatcttttc gtccggaaga acgttttccg atgatgagca
      721 cctttaaagt gctgctgtgc ggtgcggttc tgagccgtgt ggatgcgggc caggaacaac
      781 tgggccgtcg tattcattat agccagaacg atctggtgga atatagcccg gtgaccgaaa
      841 aacatctgac cgatggcatg accgtgcgtg aactgtgcag cgcggcgatt accatgagcg
      901 ataacaccgc ggcgaacctg ctgctgacga ccattggcgg tccgaaagaa ctgaccgcgt
      961 ttctgcataa catgggcgat catgtgaccc gtctggatcg ttgggaaccg gaactgaacg
     1021 aagcgattcc gaacgatgaa cgtgatacca ccatgccggc agcaatggcg accaccctgc
     1081 gtaaactgct gacgggtgag ctgctgaccc tggcaagccg ccagcaactg attgattgga
     1141 tggaagcgga taaagtggcg ggtccgctgc tgcgtagcgc gctgccggct ggctggttta
     1201 ttgcggataa aagcggtgcg ggcgaacgtg gcagccgtgg cattattgcg gcgctgggcc
     1261 cggatggtaa accgagccgt attgtggtga tttataccac cggcagccag gcgacgatgg
     1321 atgaacgtaa ccgtcagatt gcggaaattg gcgcgagcct gattaaacat tggtaaaccg
     1381 atacaattaa aggctccttt tggagccttt ttttttggac gacccttgtc cttttccgct
     1441 gcataaccct gcttcggggt cattatagcg attttttcgg tatatccatc ctttttcgca
     1501 cgatatacag gattttgcca aagggttcgt gtagactttc cttggtgtat ccaacggcgt
     1561 cagccgggca ggataggtga agtaggccca cccgcgagcg ggtgttcctt cttcactgtc
     1621 ccttattcgc acctggcggt gctcaacggg aatcctgctc tgcgaggctg gccgtaggcc
     1681 ggccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     1741 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     1801 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     1861 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     1921 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     1981 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     2041 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     2101 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     2161 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     2221 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     2281 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     2341 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     2401 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     2461 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     2521 atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     2581 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     2641 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     2701 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     2761 ggcagctgcg gtaaagctca tcagcgtggt cgtgcagcga ttcacagatg tctgcctgtt
     2821 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
     2881 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
     2941 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     3001 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     3061 tgcggcgggg gcgcgagatt gatttacccc ggttgataat cagaaaagcc ccaaaaacag
     3121 gaagattgta taagcaaata tttaaattgt aagcgttaat attttgttaa aattcgcgtt
     3181 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     3241 taaatcaaaa gaatagcccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     3301 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     3361 cccactacgt gaaccatcac ccaaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     3421 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc gaacgtggcg
     3481 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     3541 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtaaaag
     3601 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     3661 gttccactga ggcgcgccca gctgtctagg gcggcggatt tgtcctactc aggagagcgt
     3721 tcaccgacaa acaacagata aaacgaaagg cccagtcttt cgactgagcc tttcgtttta
     3781 tttgatgcct
//
