LOCUS       sequence-176803-        4310 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses NGN2, MIAT, ACTC1, and TTN gRNAs in response to 
            combinations of Cre and Flp recombinases.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4310)
  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 4310)
  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: pBW1997_pExpr-hU6-CRISPR-DECODER
FEATURES             Location/Qualifiers
     source          1..4310
                     /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=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     misc_RNA        1422..1497
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(1564..1597)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     misc_RNA        1629..1704
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    1771..1804
                     /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(1806..1839)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_RNA        1871..1946
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(2013..2046)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_RNA        2078..2153
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(2345..2364)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2410..2465
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2464..2483)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(2826..2842)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2826..2842)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2839..2861)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2850..2866
                     /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(2874..2880,2881..2898,2899..2904))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2919..2940
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      3025..3159
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3179..3313
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3216..3235)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3270..3289
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3381..3398)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3552..4140)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3632..3651)
                     /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 gcccattact cgcatccatt ctcaggctgt ctcgtctcgt ctcgcacgcg
     1321 tgaagttcct attctctaga aagtatagga acttcgataa cttcgtatag gatactttat
     1381 acgaagttat accggtcacc gggcggtggc gggggaggag ggttttagag ctagaaatag
     1441 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
     1501 tttgaattcg gtaccgctgg gagttcgtag acggaaacaa acgcagaatc caagcgcacg
     1561 cgtataactt cgtataggat actttatacg aagttatacc ggtcaccggc gcccatgaaa
     1621 ttttaatggt tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt
     1681 gaaaaagtgg caccgagtcg gtgctttttt gaattcggta ccgcactgaa ggtcctcaat
     1741 cgcactggaa acatcaaggt cggcacgcgt gaagttccta ttctctagaa agtataggaa
     1801 cttcgataac ttcgtatagc atacattata cgaagttata ccggtcaccg tggcgccctg
     1861 ccctctgctg gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac
     1921 ttgaaaaagt ggcaccgagt cggtgctttt ttgaattcgg taccctgacc tcctgccagc
     1981 aatagtaaga caacacgcaa agtcgcacgc gtataacttc gtatagcata cattatacga
     2041 agttataccg gtcaccgcct tggtgaagtc tcctttggtt ttagagctag aaatagcaag
     2101 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg
     2161 aattcggtac cgagccaact ccctttacaa cctcactcaa gtccgttaga ggcggccgcc
     2221 caggatacat agattaccac aactccgagc ccttccaccg ctagcgcact cctcaggtgc
     2281 aggctgccta tcagaaggtg gtggctggtg tggccaatgc cctggctcac aaataccact
     2341 gagatctttt tccctctgcc aaaaattatg gggacatcat gaagcccctt gagcatctga
     2401 cttctggcta ataaaggaaa tttattttca ttgcaatagt gtgttggaat tttttgtgtc
     2461 tctcactcgg aaggacatat gggagggcaa atcatttaaa acatcagaat gagtatttgg
     2521 tttagagttt ggcaacatat gcccatatgc tggctgccat gaacaaaggt tggctataaa
     2581 gaggtcatca gtatatgaaa cagccccctg ctgtccattc cttattccat agaaaagcct
     2641 tgacttgagg ttagattttt tttatatttt gttttgtgtt atttttttct ttaacatccc
     2701 taaaattttc cttacatgtt ttactagcca gatttttcct cctctcctga ctactcccag
     2761 tcatagctgt ccctcttctc ttatggagat ccctcgacct gcagcccaag cttggcgtaa
     2821 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     2881 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     2941 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gcggatccgc
     3001 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccagttcc gcccattctc
     3061 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg
     3121 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctaa
     3181 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     3241 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     3301 tcatgtctgg atccgctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     3361 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     3421 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     3481 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     3541 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     3601 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     3661 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     3721 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     3781 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     3841 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     3901 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     3961 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     4021 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     4081 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     4141 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     4201 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     4261 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
//
