LOCUS       sequence-85586-e        4856 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CFP reporter along with FF4 miRNA binding sites and 28 nt sequences.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4856)
  AUTHORS   Nissim L, Perli SD, Fridkin A, Perez-Pinera P, Lu TK
  TITLE     Multiplexed and Programmable Regulation of Gene Networks with an 
            Integrated RNA and CRISPR/Cas Toolkit in Human Cells.
  JOURNAL   Mol Cell. 2014 May 14. pii: S1097-2765(14)00355-4. doi: 
            10.1016/j.molcel.2014.04.022.
  PUBMED    24837679
REFERENCE   2  (bases 1 to 4856)
  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: "Mol Cell. 
            2014 May 14. pii: S1097-2765(14)00355-4. doi: 
            10.1016/j.molcel.2014.04.022."
COMMENT     Sequence Label: CMVp-ECFP-Triplex-28-8xmiRNA-BS-28-pA (Constru...
FEATURES             Location/Qualifiers
     source          1..4856
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(679..1470,1471..1539))
                     /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     1302..1321
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1540..1644)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1671..2126
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1758..1777)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1968..1989
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     enhancer        2315..2618
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2619..2822
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2773..2793
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2850..2859
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2856..2858,2859..2861,2862..3575)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVNKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2901..2922)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3162..3181)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3509..3530
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    3741..3760
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    3969..3988
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     primer_bind     complement(4193..4212)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4237..4284
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     complement(4605..4622)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(4776..4856,1..508))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(join(4856,1..19))
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
       61 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc
      121 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
      181 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
      241 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
      301 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
      361 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
      421 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
      481 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
      541 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
      601 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
      661 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
      721 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
      781 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
      841 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
      901 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
      961 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     1021 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     1081 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     1141 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     1201 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     1261 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     1321 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     1381 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     1441 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     1501 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     1561 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     1621 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgcgccctg
     1681 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     1741 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     1801 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     1861 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     1921 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     1981 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     2041 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     2101 taacaaaata ttaacgttta caatttccca ttcgccattc aggctgcgca actgttggga
     2161 agggcgatcg gtgcgggcct cttcgctatt acgccagccc aagctaccat gataagtaag
     2221 taatattaag gtacgggagg tacttggagg tacctagtta ttaatagtaa tcaattacgg
     2281 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
     2341 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
     2401 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
     2461 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
     2521 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt
     2581 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca
     2641 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg
     2701 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact
     2761 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag
     2821 ctggtttatg aaccgtcaga tccgagctcg ccaccatggt gaacaagggc gaggagctgt
     2881 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
     2941 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct
     3001 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctggggcg
     3061 tgcagtgctt cgcccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
     3121 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
     3181 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     3241 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac gccatcagcg
     3301 acaacgtcta tatcaccgcc gacaagcaga agaacggcat caaggccaac ttcaagatcc
     3361 gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca
     3421 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag tccaagctga
     3481 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
     3541 ggatcactct cggcatggac gagctgtaca agtaaaccgg tgattcgtca gtagggttgt
     3601 aaaggttttt cttttcctga gaaaacaacc ttttgttttc tcaggttttg ctttttggcc
     3661 tttccctagc tttaaaaaaa aaaagcaaaa ctcaccgagg cagttccata ggatggcaag
     3721 atcctggtat cggtctgcga gttcactgcc gtataggcag ctaagaaacc gcttgaagtc
     3781 tttaattaaa ccgcttgaag tctttaatta aaccgcttga agtctttaat taaaccgctt
     3841 gaagtcttta attaaacctc tggccacatc ggttcctgct ccgcttgaag tctttaatta
     3901 aaccgcttga agtctttaat taaaccgctt gaagtcttta attaaaccgc ttgaagtctt
     3961 taattaaagt tcactgccgt ataggcagct aagaaacccg ggtaagcggg actctggggt
     4021 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     4081 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     4141 agcgcgggga tctcatgctg gagttcttcg cccaccctag ggggaggcta actgaaacac
     4201 ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga cagaataaaa
     4261 cgcacggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc tggcactctg
     4321 tcgatacccc accgagacgc cattggggcc aatacgcccg cgtttcttcc ttttccccac
     4381 cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc
     4441 ctgccatagc ctcagctcga gtatgtcgac cgatgccctt gagagccttc aacccagtca
     4501 gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact gtcttcttta
     4561 tcatgcaact cgtaggacag gtgccggcag cgctcttccg cttcctcgct cactgactcg
     4621 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     4681 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     4741 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     4801 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actata
//
