LOCUS       sequence-123992-        3520 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains the sgRNA backbone sequence (tracrRNA, 82 bp) and is used 
            as DNA template to amplify a specific sgRNA using a forward primer 
            with the protospacer sequence for gene targeting..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3520)
  AUTHORS   Lander N, Li ZH, Niyogi S, Docampo R
  TITLE     CRISPR/Cas9-Induced Disruption of Paraflagellar Rod Protein 1 and 2 
            Genes in Trypanosoma cruzi Reveals Their Role in Flagellar 
            Attachment.
  JOURNAL   MBio. 2015 Jul 21;6(4). pii: e01012-15. doi: 10.1128/mBio.01012-15.
  PUBMED    26199333
REFERENCE   2  (bases 1 to 3520)
  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: "MBio."; 
            date: "2015-07-21"; volume: "6(4). pii: e01012-15. doi"; pages: " 
            10.1128/mBio.01012-15"
COMMENT     Sequence Label: pUC_sgRNA
FEATURES             Location/Qualifiers
     source          1..3520
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(6..23)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6..22)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(15..37)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      235..690
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(322..341)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     532..553
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(707..726)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(886..904)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        972..1076
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1077..1145,1146..1937)
                     /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     complement(1295..1314)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2108..2696
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2597..2616
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2850..2867
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2984..3005
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3020..3025,3026..3043,3044..3050)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3058..3074
                     /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)."
     primer_bind     3063..3085
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3082..3098
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3082..3098
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_RNA        complement(3355..3430)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
       61 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
      121 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt
      181 ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc
      241 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
      301 ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
      361 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
      421 tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc
      481 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
      541 tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat ttataaggga
      601 ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
      661 attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg
      721 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
      781 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtcaacggg agctgcatgt
      841 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgacccg aaagggcctc gtgatacgcc
      901 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
      961 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     1021 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     1081 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     1141 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     1201 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     1261 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     1321 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     1381 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     1441 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     1501 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     1561 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     1621 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     1681 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     1741 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt ggttctcgcg
     1801 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     1861 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     1921 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     1981 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     2041 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     2101 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     2161 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     2221 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     2281 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     2341 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     2401 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     2461 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     2521 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     2581 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     2641 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     2701 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     2761 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     2821 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     2881 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     2941 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca
     3001 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
     3061 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc aagctccttc
     3121 ctcttccagc ccttcctctt tcgggcggca gcaagatccc ttctgatcta aagtccaacc
     3181 ggcgagagtg aacggcactg cgcttggttt agttgtcaag agagaataac tctcggacgc
     3241 ggaaggattc gtagcgtata tcgcctattg cggaagcgta accaagtctt acgaccccaa
     3301 aagaggttga ctgaactggg acgcggcact ggcgcaaagc ttggatccaa aaaagcaccg
     3361 actcggtgcc actttttcaa gttgataacg gactagcctt attttaactt gctatttcta
     3421 gctctaaaac ccatggaatt cgtgtcgcat gaataacggg cagttatcaa atgagcacgg
     3481 cgacagcctg tgaaagtcct ctccactgac tgtagcctcc
//
