LOCUS       sequence-401593-        4696 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a non-coding transcript expressed from the EF1a promoter 
            that has a 3' UTR containing a MALAT1 triplex, Csy4 binding sites 
            (""28""), and a cloning site for a Cas9 gRNA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4696)
  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 4696)
  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: EF1a-Triplex-28-M13-28-pA
FEATURES             Location/Qualifiers
     source          1..4696
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(182..973,974..1042))
                     /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     805..824
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1043..1147)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1174..1629
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1261..1280)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1471..1492
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1762..2940
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1993..2931
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2888..2908
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    3110..3129
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     primer_bind     3145..3161
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3145..3161
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_RNA        3170..3245
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    3254..3273
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     primer_bind     complement(3536..3555)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3580..3627
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     complement(3948..3965)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(4119..4696,1..11))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4199..4218)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
       61 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
      121 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
      181 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
      241 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
      301 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
      361 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
      421 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
      481 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
      541 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
      601 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
      661 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
      721 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
      781 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
      841 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
      901 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
      961 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     1021 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     1081 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     1141 ttccgcgcac atttccccga aaagtgccac ctgacgcgcc ctgtagcggc gcattaagcg
     1201 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     1261 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     1321 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     1381 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     1441 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     1501 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     1561 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     1621 ttacaatttg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
     1681 cctcttcgct attacgccag cccaagctac catgataagt aagtaatatt aaggtacggg
     1741 aggtacttgg aggtaccaat tggctccggt gcccgtcagt gggcagagcg cacatcgccc
     1801 acagtccccg agaagttggg gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg
     1861 cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gcctttttcc cgagggtggg
     1921 ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc
     1981 gccagaacac aggtaagtgc cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat
     2041 ggcccttgcg tgccttgaat tacttccacc tggctgcagt acgtgattct tgatcccgag
     2101 cttcgggttg gaagtgggtg ggagagttcg aggccttgcg cttaaggagc cccttcgcct
     2161 cgtgcttgag ttgaggcctg gcctgggcgc tggggccgcc gcgtgcgaat ctggtggcac
     2221 cttcgcgcct gtctcgctgc tttcgataag tctctagcca tttaaaattt ttgatgacct
     2281 gctgcgacgc tttttttctg gcaagatagt cttgtaaatg cgggccaaga tctgcacact
     2341 ggtatttcgg tttttggggc cgcgggcggc gacggggccc gtgcgtccca gcgcacatgt
     2401 tcggcgaggc ggggcctgcg agcgcggcca ccgagaatcg gacgggggta gtctcaagct
     2461 ggccggcctg ctctggtgcc tggcctcgcg ccgccgtgta tcgccccgcc ctgggcggca
     2521 aggctggccc ggtcggcacc agttgcgtga gcggaaagat ggccgcttcc cggccctgct
     2581 gcagggagct caaaatggag gacgcggcgc tcgggagagc gggcgggtga gtcacccaca
     2641 caaaggaaaa gggcctttcc gtcctcagcc gtcgcttcat gtgactccac ggagtaccgg
     2701 gcgccgtcca ggcacctcga ttagttctcg agcttttgga gtacgtcgtc tttaggttgg
     2761 ggggaggggt tttatgcgat ggagtttccc cacactgagt gggtggagac tgaagttagg
     2821 ccagcttggc acttgatgta attctccttg gaatttgccc tttttgagtt tggatcttgg
     2881 ttcattctca agcctcagac agtggttcaa agtttttttc ttccatttca ggtgtcgtga
     2941 ggtctagacg attcgtcagt agggttgtaa aggtttttct tttcctgaga aaacaacctt
     3001 ttgttttctc aggttttgct ttttggcctt tccctagctt taaaaaaaaa aaagcaaaac
     3061 tcaccgaggc agttccatag gatggcaaga tcctggtatt ggtctgcgag ttcactgccg
     3121 tataggcagc taagaaagca ggtgcaggaa acagctatga ccacctgctg ttttagagct
     3181 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
     3241 ggtgcttttt ttcgttcact gccgtatagg cagctaagaa acaaacagga atcgaatgca
     3301 accggcgcag gaacactgcc agcgcatcaa ccccgggatg ctagctaagc gggactctgg
     3361 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
     3421 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     3481 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc tagggggagg ctaactgaaa
     3541 cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata
     3601 aaacgcacgg tgttgggtcg tttgttcata aacgcggggt tcggtcccag ggctggcact
     3661 ctgtcgatac cccaccgaga cgccattggg gccaatacgc ccgcgtttct tccttttccc
     3721 caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag
     3781 gccctgccat agcctcagct cgagtatgtc gaccgatgcc cttgagagcc ttcaacccag
     3841 tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg actgtcttct
     3901 ttatcatgca actcgtagga caggtgccgg cagcgctctt ccgcttcctc gctcactgac
     3961 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4021 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4081 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4141 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4201 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4261 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4321 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4381 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4441 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4501 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     4561 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     4621 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4681 attacgcgca gaaaaa
//
