LOCUS       sequence-120214-        3591 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient expression of an ""INT"" construct_bearing three PP7 
            Stem-loops, targeting the GLuc reporter, in mammalian cells. CMV/3' 
            Box expression backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3591)
  AUTHORS   Shechner DM, Hacisuleyman E, Younger ST, Rinn JL
  TITLE     Multiplexable, locus-specific targeting of long RNAs with 
            CRISPR-Display.
  JOURNAL   Nat Methods. 2015 Jul;12(7):664-70. doi: 10.1038/nmeth.3433. Epub 
            2015 Jun 1.
  PUBMED    26030444
REFERENCE   2  (bases 1 to 3591)
  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"; doi: 
            "10.1038/nmeth.3433"; journalName: "Nat Methods"; date: "2015-07"; 
            volume: "12"; issue: "7"; pages: "664-70"
COMMENT     Sequence Label: pCMV/3' Box_(GLuc)_INT
FEATURES             Location/Qualifiers
     source          1..3591
                     /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
     primer_bind     1034..1052
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1084..1106)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1200..1219
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1413..1435
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1427..1444
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1428..1444
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        1463..1841
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1842..2045
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1996..2016
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     complement(2431..2447)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2431..2447)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2444..2466)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2455..2471
                     /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(2479..2485,2486..2503,2504..2509))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2524..2545
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2662..2679)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2833..3421)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2913..2932)
                     /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 tgacgtctaa gaaaccatta ttatcatgac
     1021 attaacctat aaaaataggc gtatcacgag gccctttgcg cgtttcggtg atgacggtga
     1081 aaacctctga cacatgcagc tcccggtcac agcttgtctg taagcggatg ccgggagcag
     1141 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc
     1201 ggcatcagag cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg
     1261 cgtaaggaga aaataccgca tcaggcgcca ttcgccattc aggctgcgca actgttggga
     1321 agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc
     1381 aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc
     1441 cagtgaattc gagctcggta ctacattgat tattgactag ttattaatag taatcaatta
     1501 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
     1561 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
     1621 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
     1681 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
     1741 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
     1801 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
     1861 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
     1921 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
     1981 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
     2041 gagctctctg gctaactaga gaacccactg cttactggcg atctagatac gactcactat
     2101 gttttagagc tacgaatacg agggagcaga cgatatggcg tcgctcctct ccacgagagc
     2161 atatgggctc cgtggtctgc agcagacgat atggcgtcgc tgctctcgta gatgccatat
     2221 ggggcactac gtctccagca gacgatatgg cgtcgctggc tcgtattcgt agcaagttaa
     2281 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttactttctg
     2341 gagtttcaaa agtagactgt acgctaaggg tcatatcttt ttttgtttgg tttgtgtctt
     2401 ggttggcgtc ttaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     2461 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     2521 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     2581 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     2641 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     2701 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     2761 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     2821 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     2881 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     2941 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     3001 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     3061 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     3121 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     3181 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     3241 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     3301 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     3361 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     3421 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     3481 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     3541 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca g
//
