LOCUS       sequence-120213-        3769 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient expression of the ""TOP2"" construct, 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 3769)
  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 3769)
  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)_TOP2
FEATURES             Location/Qualifiers
     source          1..3769
                     /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"
     misc_RNA        2435..2510
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(2609..2625)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2609..2625)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2622..2644)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2633..2649
                     /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(2657..2663,2664..2681,2682..2687))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2702..2723
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2840..2857)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3011..3599)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3091..3110)
                     /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 gaattgcggg aaaggggtca
     2101 acagccgttc agtaccaagt ctcaggggaa actttgagat ggccttgcaa agggtatggt
     2161 aataagctga cggacatggt cctaacacgc agccaagtcc taagtcaaca gtttttggag
     2221 cagacgatat ggcgtcgctc ctttttccag cagagcatat gggctccgtg gtttttccag
     2281 cagacgatat ggcgtcgctg gtttttggag cagagcatat gggctcgctc ctttttgcag
     2341 cagacgatat ggcgtcgctg ctttttctgt tgatatggat gcagttcatc ttcaacatcc
     2401 ctcctctaaa cccgatctag atacgactca ctatgtttta gagctagaaa tagcaagtta
     2461 aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc actttctgga
     2521 gtttcaaaag tagactgtac gctaagggtc atatcttttt ttgtttggtt tgtgtcttgg
     2581 ttggcgtctt aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     2641 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     2701 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     2761 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     2821 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     2881 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     2941 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     3001 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     3061 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     3121 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     3181 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     3241 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     3301 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     3361 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     3421 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     3481 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     3541 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     3601 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     3661 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     3721 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacag
//
