LOCUS       sequence-207490-        3130 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient expression of an ""INT"" construct [bearing six minimal 
            Puf1 stem-loops; 'INT' design: Shechner et al., Nature Methods 
            2015], in mammalian cells, U6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3130)
  AUTHORS   Maass PG, Barutcu AR, Shechner DM, Weiner CL, Mele M, Rinn JL
  TITLE     Spatiotemporal allele organization by allele-specific CRISPR 
            live-cell imaging (SNP-CLING).
  JOURNAL   Nat Struct Mol Biol. 2018 Jan 8. pii: 10.1038/s41594-017-0015-3. 
            doi: 10.1038/s41594-017-0015-3.
  PUBMED    29343869
REFERENCE   2  (bases 1 to 3130)
  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 Struct 
            Mol Biol. 2018 Jan 8. pii: 10.1038/s41594-017-0015-3. doi: 
            10.1038/s41594-017-0015-3."
COMMENT     Sequence Label: pU6_T7_INT[6xminimal_Puf1]
FEATURES             Location/Qualifiers
     source          1..3130
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(23..814,815..883))
                     /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     646..665
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(884..988)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1056..1074
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1112..1134)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1234..1253
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1447..1469
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1461..1478
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1462..1478
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1642..1661
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1992..2008)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1992..2008)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2005..2027)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2016..2032
                     /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(2040..2046,2047..2064,2065..2070))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2085..2106
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2223..2240)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2394..2982)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2474..2493)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
       61 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
      121 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
      181 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
      241 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
      301 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
      361 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
      421 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
      481 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
      541 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
      601 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
      661 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
      721 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
      781 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
      841 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
      901 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
      961 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct
     1021 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
     1081 gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg
     1141 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
     1201 gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag
     1261 tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc
     1321 gccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
     1381 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
     1441 ggttttccca gtcacgacgt tgtaaaacga cggccagtgt ttcccatgat tccttcatat
     1501 ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca
     1561 aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt
     1621 ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat
     1681 ttcttggctt tatatatctt gtggaaagga cgaaacaccg atctagatac gactcactat
     1741 gtttaagagc tatgctgcga atacgagtct attgtatata tctattgtat atatctattg
     1801 tatatatcta ttgtatatat ctattgtata tatctattgt atatatctct cgtattcgca
     1861 gcatagcaag tttaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg
     1921 tgcttttttt cttaattaag tctagagtcg actgtttaaa cctgcaggca tgcaagcttg
     1981 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     2041 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     2101 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     2161 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     2221 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     2281 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     2341 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     2401 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     2461 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     2521 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     2581 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     2641 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     2701 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     2761 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     2821 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     2881 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     2941 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     3001 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     3061 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     3121 taaagtatat
//
