LOCUS       sequence-401605-        4279 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, 
            dCas12a DRs, and cloning site for crRNA to activate downstream 
            targets..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4279)
  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 4279)
  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-DR-M13-DR-pA
FEATURES             Location/Qualifiers
     source          1..4279
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(190..981,982..1050))
                     /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     813..832
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1051..1155)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1182..1637
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1269..1288)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1479..1500
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    1768..1816
                     /note="synthetic polyadenylation signal"
     misc_feature    1830..1921
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     1870..1889
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     primer_bind     1935..1951
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2021..2725
                     /codon_start=1
                     /product="Phialidium yellow fluorescent proteim"
                     /label=PhiYFP
                     /note="mammalian codon-optimized"
                     /translation="MSSGALLFHGKIPYVVEMEGDVDGHTFSIRGKGYGDASVGKVDAQ
                     FICTTGDVPVPWSTLVTTLTYGAQCFAKYGPELKDFYKSCMPDGYVQERTITFEGDGNF
                     KTRAEVTFENGSVYNRVKLNGQGFKKDGHVLGKNLEFNFTPHCLYIWGDQANHGLKSAF
                     KICHEIAGSKGDFIVADHTQMNTPIGGGPVHVPEYHHMSYHVKLSKDVTDHRDNMSLTE
                     TVRAVDCRKTYL"
     primer_bind     2929..2945
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3203..3222)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3247..3294
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     complement(3539..3556)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(3710..4279,1..19))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3790..3809)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
       61 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
      121 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
      181 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
      241 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
      301 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
      361 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
      421 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
      481 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
      541 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
      601 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
      661 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
      721 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
      781 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
      841 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
      901 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
      961 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     1021 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     1081 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     1141 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgcgccct gtagcggcgc
     1201 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     1261 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     1321 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     1381 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     1441 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     1501 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     1561 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     1621 attaacgctt acaatttgcc attcgccatt caggctgcgc aactgttggg aagggcgatc
     1681 ggtgcgggcc tcttcgctat tacgccagcc caagctacca tgataagtaa gtaatattaa
     1741 ggtacgggag gtacttggag cggccgcaat aaaatatctt tattttcatt acatctgtgt
     1801 gttggttttt tgtgtgaatc gatagtacta acatacgctc tccatcaaaa caaaacgaaa
     1861 caaaacaaac tagcaaaata ggctgtcccc agtgcaagtg caggtgccag aacatttctc
     1921 tatcgatagg atcccaggaa acagctatga cgagctctct agcggggggc tataaaaggg
     1981 ggtgggggcg ttcgtcctgc tatctagcgt cgcgttgacc atgagcagcg gcgccctgct
     2041 gttccacggc aagatcccct acgtggtgga gatggagggc gatgtggatg gccacacctt
     2101 cagcatccgc ggtaagggct acggcgatgc cagcgtgggc aaggtggatg cccagttcat
     2161 ctgcaccacc ggcgatgtgc ccgtgccctg gagcaccctg gtgaccaccc tgacctacgg
     2221 cgcccagtgc ttcgccaagt acggccccga gctgaaggat ttctacaaga gctgcatgcc
     2281 cgatggctac gtgcaggagc gcaccatcac cttcgagggc gatggcaatt tcaagacccg
     2341 cgccgaggtg accttcgaga atggcagcgt gtacaatcgc gtgaagctga atggccaggg
     2401 cttcaagaag gatggccacg tgctgggcaa gaatctggag ttcaatttca ccccccactg
     2461 cctgtacatc tggggcgatc aggccaatca cggcctgaag agcgccttca agatctgcca
     2521 cgagatcgcc ggcagcaagg gcgatttcat cgtggccgat cacacccaga tgaatacccc
     2581 catcggcggc ggccccgtgc acgtgcccga gtaccaccac atgagctacc acgtgaagct
     2641 gagcaaggat gtgaccgatc accgcgataa tatgagcctg acggagaccg tgcgcgccgt
     2701 ggattgccgc aagacctacc tgtaaggccg gcctaaaccg gtgattcgtc agtagggttg
     2761 taaaggtttt tcttttcctg agaaaacaac cttttgtttt ctcaggtttt gctttttggc
     2821 ctttccctag ctttaaaaaa aaaaaagcaa aactcaccga ggcagttcca taggatggca
     2881 agatcctggt attggtctgc gaaatttcta ctcttgtaga ttgagacgca ggaaacagct
     2941 atgaccgtct ctaatttcta ctcttgtaga ttctagaaaa gaattcgata tcaagcttcc
     3001 cgggatgcta gctaagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     3061 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     3121 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     3181 cccaccctag ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg
     3241 ctatgacggc aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt gttccaattg
     3301 cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa
     3361 cctctacaaa tgtggtaaaa tcgataagga tcgagtatgt cgaccgatgc ccttgagagc
     3421 cttcaaccca gtcagctcct tccggtgggc gcggggcatg actatcgtcg ccgcacttat
     3481 gactgtcttc tttatcatgc aactcgtagg acaggtgccg gcagcgctct tccgcttcct
     3541 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     3601 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     3661 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     3721 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     3781 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     3841 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     3901 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     3961 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     4021 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     4081 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4141 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4201 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     4261 gcaagcagca gattacgcg
//
