LOCUS       sequence-396680-        4186 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian transfections; 8xCTS-ECFP reporter construct 4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4186)
  AUTHORS   Pelea O, Mayes S, Ferry QRV, Fulga TA, Sauka-Spengler T
  TITLE     Specific Modulation of CRISPR Transcriptional Activators through 
            RNA-Sensing Guide RNAs in Mammalian Cells and Zebrafish Embryos
  JOURNAL   bioRxiv 2023.05.08.539738
REFERENCE   2  (bases 1 to 4186)
  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: "bioRxiv 
            2023.05.08.539738"
COMMENT     Sequence Label: gRNA4 _8xCTS-ECFP-pA
FEATURES             Location/Qualifiers
     source          1..4186
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(448..1239,1240..1308))
                     /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     1071..1090
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1309..1413)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1440..1895
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1527..1546)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1737..1758
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    2087..2178
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     2127..2146
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     CDS             join(2585..2587,2588..2590,2591..3304)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2630..2651)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2891..2910)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3238..3259
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3330..3451
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3341..3360)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3395..3414
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3704..3721)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(3875..4186,1..277))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3955..3974)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
       61 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
      121 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
      181 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
      241 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
      301 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
      361 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
      421 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
      481 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
      541 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
      601 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
      661 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
      721 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
      781 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
      841 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
      901 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
      961 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     1021 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     1081 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     1141 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     1201 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     1261 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     1321 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     1381 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     1441 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     1501 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     1561 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     1621 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     1681 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     1741 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     1801 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     1861 cgcgaatttt aacaaaatat taacgcttac aatttgccat tcgccattca ggctgcgcaa
     1921 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagccca agctaccatg
     1981 ataagtaagt aatattaagg tacgggaggt acttggagcg gccgcaataa atatctttat
     2041 tttcattaca tctgtgtgtt ggttttttgt gtgaatcgat agtactaaca tacgctctcc
     2101 atcaaaacaa aacgaaacaa aacaaactag caaaataggc tgtccccagt gcaagtgcag
     2161 gtgccagaac atttctctat cgatagctag cactagtccc tcctgctagt cattacctgg
     2221 ttaatatttt catagccgtc ctgctagtca ttacctggga gctcctactg accctactag
     2281 tccctcctgc tagtcattac ctggttaata ttttcatagc cgtcctgcta gtcattacct
     2341 gggagctctc taggggccca ctagtccctc ctgctagtca ttacctggtt aatattttca
     2401 tagccgtcct gctagtcatt acctgggagc tcctactgac cctactagtc cctcctgcta
     2461 gtcattacct ggttaatatt ttcatagccg tcctgctagt cattacctgg gagctctcta
     2521 gactagcggg gggctataaa agggggtggg ggcgttcgtc ctgctatcta gcgtcgcgtt
     2581 gaccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
     2641 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
     2701 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     2761 caccctcgtg accaccctga cctggggcgt gcagtgcttc gcccgctacc ccgaccacat
     2821 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     2881 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     2941 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     3001 gcacaagctg gagtacaacg ccatcagcga caacgtctat atcaccgccg acaagcagaa
     3061 gaacggcatc aaggccaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     3121 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     3181 ccactacctg agcacccagt ccaagctgag caaagacccc aacgagaagc gcgatcacat
     3241 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     3301 gtaaggccgg ccgcttcgag cagacatgat aagatacatt gatgagtttg gacaaaccac
     3361 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
     3421 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt
     3481 tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg
     3541 taaaatcgat aaggatcgag tatgtcgacc gatgcccttg agagccttca acccagtcag
     3601 ctccttccgg tgggcgcggg gcatgactat cgtcgccgca cttatgactg tcttctttat
     3661 catgcaactc gtaggacagg tgccggcagc gctcttccgc ttcctcgctc actgactcgc
     3721 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     3781 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     3841 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     3901 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     3961 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     4021 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     4081 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     4141 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttga
//
