LOCUS       sequence-243238-        3230 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid expressing sgRNA2 (TACCACATTTGTAGAGGTT).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3230)
  AUTHORS   Rui Y, Varanasi M, Mendes S, Yamagata HM, Wilson DR, Green JJ
  TITLE     Poly(Beta-Amino Ester) Nanoparticles Enable Nonviral Delivery of 
            CRISPR-Cas9 Plasmids for Gene Knockout and Gene Deletion.
  JOURNAL   Mol Ther Nucleic Acids. 2020 Apr 21;20:661-672. doi: 
            10.1016/j.omtn.2020.04.005.
  PUBMED    32380416
REFERENCE   2  (bases 1 to 3230)
  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: "Mol Ther 
            Nucleic Acids."; date: "2020-04-21"; volume: "20:661-672. doi"; 
            pages: " 10.1016/j.omtn.2020.04.005"
COMMENT     Sequence Label: pPTG-GG-sg2+sg3
FEATURES             Location/Qualifiers
     source          1..3230
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(294..1085,1086..1154))
                     /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     917..936
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1155..1259)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1390..1638
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1390..1410
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1561..1580
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1741..1816
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_RNA        1914..1989
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(2018..2034)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2018..2034)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2031..2053)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2042..2058
                     /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(2066..2072,2073..2090,2091..2096))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2111..2132
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2191..2386
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2237..2372
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2392..2526
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2429..2448)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2483..2502
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2594..2611)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(2765..3230,1..123))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2845..2864)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
       61 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
      121 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
      181 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
      241 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
      301 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
      361 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
      421 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
      481 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
      541 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
      601 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
      661 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
      721 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
      781 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
      841 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
      901 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
      961 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     1021 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     1081 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     1141 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     1201 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     1261 acatttcccc gaaaagtgcc acctggtcga cattgattat tgactagtag tggagaattc
     1321 tgtacaaaaa agcaggcttt aaaggaacca attcagtcga ctggatccgg taccaaggtc
     1381 gggcaggaag agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
     1441 gttagagaga taattagaat taatttgact gtaaacacaa agatattagt acaaaatacg
     1501 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
     1561 gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg
     1621 tggaaaggac gaaacacctc tagaacaaag caccagtggt ctagtggtag aatagtaccc
     1681 tgccacggta cagacccggg ttcgattccc ggctggtgca gaacctctac aaatgtggta
     1741 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
     1801 ggcaccgagt cggtgcaaca aagcaccagt ggtctagtgg tagaatagta ccctgccacg
     1861 gtacagaccc gggttcgatt cccggctggt gcagcaatgt atcttatcat gtcgttttag
     1921 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
     1981 agtcggtgct ttttttttta agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     2041 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     2101 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     2161 agtcgggaaa cctgtcgtgc cagcggatcc gcatctcaat tagtcagcaa ccatagtccc
     2221 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     2281 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt
     2341 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc taacttgttt
     2401 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     2461 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     2521 tggatccgct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     2581 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     2641 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     2701 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     2761 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     2821 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     2881 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     2941 gaagcgtggc gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc
     3001 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     3061 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     3121 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     3181 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
//
