LOCUS       sequence-177166-        3651 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PV3 gRNA entry vector for CRISPR decoder.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3651)
  AUTHORS   Weinberg BH, Pham NT, Caraballo LD, Lozanoski T, Engel A, Bhatia S, 
            Wong WW
  TITLE     Large-scale design of robust genetic circuits with multiple inputs 
            and outputs for mammalian cells.
  JOURNAL   Nat Biotechnol. 2017 Mar 27. doi: 10.1038/nbt.3805.
  PUBMED    28346402
REFERENCE   2  (bases 1 to 3651)
  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: "Nat 
            Biotechnol. 2017 Mar 27. doi: 10.1038/nbt.3805."
COMMENT     Sequence Label: pBW1943_ hU6-PV3-FRT-loxP-BbsI-ENTRY-gRNA
FEATURES             Location/Qualifiers
     source          1..3651
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(1..792,793..861))
                     /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     624..643
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(862..966)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1017..1265
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1017..1037
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1188..1207
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    1322..1355
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     protein_bind    complement(1357..1390)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_RNA        1419..1494
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1686..1705)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    1751..1806
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1805..1824)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(2167..2183)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2167..2183)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2180..2202)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2191..2207
                     /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(2215..2221,2222..2239,2240..2245))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2260..2281
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      2366..2500
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2520..2654
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2557..2576)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2611..2630
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2722..2739)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2893..3481)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2973..2992)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
       61 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
      121 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
      181 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
      241 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
      301 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
      361 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
      421 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
      481 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
      541 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
      601 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
      661 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
      721 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
      781 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
      841 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
      901 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
      961 tccgcgcaca tttccccgaa aagtgccacc tgggtcgaca ttgattattg actagtgagg
     1021 gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt agagagataa
     1081 ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt
     1141 aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc
     1201 ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa
     1261 acaccggcgc gccgcactga aggtcctcaa tcgcactgga aacatcaagg tcggcacgcg
     1321 tgaagttcct attctctaga aagtatagga acttcgataa cttcgtatag catacattat
     1381 acgaagttat accggtcacc gggtcttcga gaagacctgt tttagagcta gaaatagcaa
     1441 gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt
     1501 gaattcggta ccctgacctc ctgccagcaa tagtaagaca acacgcaaag tcgcggccgc
     1561 ccaggataca tagattacca caactccgag cccttccacc gctagcgcac tcctcaggtg
     1621 caggctgcct atcagaaggt ggtggctggt gtggccaatg ccctggctca caaataccac
     1681 tgagatcttt ttccctctgc caaaaattat ggggacatca tgaagcccct tgagcatctg
     1741 acttctggct aataaaggaa atttattttc attgcaatag tgtgttggaa ttttttgtgt
     1801 ctctcactcg gaaggacata tgggagggca aatcatttaa aacatcagaa tgagtatttg
     1861 gtttagagtt tggcaacata tgcccatatg ctggctgcca tgaacaaagg ttggctataa
     1921 agaggtcatc agtatatgaa acagccccct gctgtccatt ccttattcca tagaaaagcc
     1981 ttgacttgag gttagatttt ttttatattt tgttttgtgt tatttttttc tttaacatcc
     2041 ctaaaatttt ccttacatgt tttactagcc agatttttcc tcctctcctg actactccca
     2101 gtcatagctg tccctcttct cttatggaga tccctcgacc tgcagcccaa gcttggcgta
     2161 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     2221 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     2281 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agcggatccg
     2341 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccagttc cgcccattct
     2401 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     2461 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagcta
     2521 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     2581 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     2641 atcatgtctg gatccgctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     2701 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     2761 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     2821 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     2881 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     2941 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     3001 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     3061 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     3121 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     3181 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     3241 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     3301 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     3361 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     3421 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     3481 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     3541 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     3601 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca g
//
