LOCUS       sequence-401599-        4403 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes PhiYFP downstream of a cloning site for constructing a 
            synthetic Cas-targeted promoter. 3' UTR has a MALAT1 triplex, Csy4 
            28nt sites, and cloning site for gRNA to activate downstream 
            targets..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4403)
  AUTHORS   Kang H, Fitch JC, Varghese RP, Thorne CA, Cusanovich DA
  TITLE     Optimization of a Cas12a-Driven Synthetic Gene Regulatory Network 
            System.
  JOURNAL   ACS Synth Biol. 2025 May 16;14(5):1732-1744. doi: 
            10.1021/acssynbio.5c00084. Epub 2025 May 2.
  PUBMED    40316310
REFERENCE   2  (bases 1 to 4403)
  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: 
            "10.1021/acssynbio.5c00084"; journalName: "ACS Synth Biol"; date: 
            "2025-05-16- 16"; volume: "14"; issue: "5"; pages: "1732-1744"
COMMENT     Sequence Label: Px-PhiYFP-triplex-28-M13-28-pA
FEATURES             Location/Qualifiers
     source          1..4403
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(174..965,966..1034))
                     /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     797..816
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1035..1139)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1166..1621
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1253..1272)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1463..1484
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    1752..1800
                     /note="synthetic polyadenylation signal"
     misc_feature    1814..1905
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     1854..1873
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     primer_bind     1919..1935
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2005..2709
                     /codon_start=1
                     /product="Phialidium yellow fluorescent proteim"
                     /label=PhiYFP
                     /note="mammalian codon-optimized"
                     /translation="MSSGALLFHGKIPYVVEMEGDVDGHTFSIRGKGYGDASVGKVDAQ
                     FICTTGDVPVPWSTLVTTLTYGAQCFAKYGPELKDFYKSCMPDGYVQERTITFEGDGNF
                     KTRAEVTFENGSVYNRVKLNGQGFKKDGHVLGKNLEFNFTPHCLYIWGDQANHGLKSAF
                     KICHEIAGSKGDFIVADHTQMNTPIGGGPVHVPEYHHMSYHVKLSKDVTDHRDNMSLTE
                     TVRAVDCRKTYL"
     protein_bind    2887..2906
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     primer_bind     2921..2937
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_RNA        2945..3020
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    3029..3048
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     primer_bind     complement(3311..3330)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3355..3402
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     complement(3647..3664)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(3818..4403,1..3))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3898..3917)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
       61 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
      121 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
      181 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
      241 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
      301 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
      361 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
      421 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
      481 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
      541 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
      601 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
      661 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
      721 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
      781 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
      841 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
      901 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
      961 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     1021 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     1081 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     1141 acatttcccc gaaaagtgcc acctgacgcg ccctgtagcg gcgcattaag cgcggcgggt
     1201 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     1261 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     1321 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     1381 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     1441 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     1501 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     1561 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     1621 tgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg
     1681 ctattacgcc agcccaagct accatgataa gtaagtaata ttaaggtacg ggaggtactt
     1741 ggagcggccg caataaaata tctttatttt cattacatct gtgtgttggt tttttgtgtg
     1801 aatcgatagt actaacatac gctctccatc aaaacaaaac gaaacaaaac aaactagcaa
     1861 aataggctgt ccccagtgca agtgcaggtg ccagaacatt tctctatcga taggatccca
     1921 ggaaacagct atgacgagct ctctagcggg gggctataaa agggggtggg ggcgttcgtc
     1981 ctgctatcta gcgtcgcgtt gaccatgagc agcggcgccc tgctgttcca cggcaagatc
     2041 ccctacgtgg tggagatgga gggcgatgtg gatggccaca ccttcagcat ccgcggtaag
     2101 ggctacggcg atgccagcgt gggcaaggtg gatgcccagt tcatctgcac caccggcgat
     2161 gtgcccgtgc cctggagcac cctggtgacc accctgacct acggcgccca gtgcttcgcc
     2221 aagtacggcc ccgagctgaa ggatttctac aagagctgca tgcccgatgg ctacgtgcag
     2281 gagcgcacca tcaccttcga gggcgatggc aatttcaaga cccgcgccga ggtgaccttc
     2341 gagaatggca gcgtgtacaa tcgcgtgaag ctgaatggcc agggcttcaa gaaggatggc
     2401 cacgtgctgg gcaagaatct ggagttcaat ttcacccccc actgcctgta catctggggc
     2461 gatcaggcca atcacggcct gaagagcgcc ttcaagatct gccacgagat cgccggcagc
     2521 aagggcgatt tcatcgtggc cgatcacacc cagatgaata cccccatcgg cggcggcccc
     2581 gtgcacgtgc ccgagtacca ccacatgagc taccacgtga agctgagcaa ggatgtgacc
     2641 gatcaccgcg ataatatgag cctgacggag accgtgcgcg ccgtggattg ccgcaagacc
     2701 tacctgtaag gccggcctaa accggtgatt cgtcagtagg gttgtaaagg tttttctttt
     2761 cctgagaaaa caaccttttg ttttctcagg ttttgctttt tggcctttcc ctagctttaa
     2821 aaaaaaaaaa gcaaaactca ccgaggcagt tccataggat ggcaagatcc tggtattggt
     2881 ctgcgagttc actgccgtat aggcagctaa gaaagagacg caggaaacag ctatgaccgt
     2941 ctctgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa
     3001 aagtggcacc gagtcggtgc tttttttcgt tcactgccgt ataggcagct aagaaacaaa
     3061 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaaccccg ggatgctagc
     3121 taagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag
     3181 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     3241 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg
     3301 ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa
     3361 taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt tccaattgca ttcattttat
     3421 gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg
     3481 tggtaaaatc gataaggatc gagtatgtcg accgatgccc ttgagagcct tcaacccagt
     3541 cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt
     3601 tatcatgcaa ctcgtaggac aggtgccggc agcgctcttc cgcttcctcg ctcactgact
     3661 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     3721 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     3781 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     3841 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     3901 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     3961 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     4021 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     4081 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     4141 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     4201 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     4261 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     4321 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     4381 ttacgcgcag aaaaaaagga tct
//
