LOCUS       sequence-484258-        5134 bp    DNA     circular SYN 21-AUG-2025
DEFINITION  Mammalian vector for transient expression of theophylline-responsive
            MR-scRNA with mCherry transfection marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5134)
  TITLE     Metabolite-responsive scaffold RNAs for dynamic CRISPR 
            transcriptional regulation
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5134)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 21, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pHS-MRscRNA
FEATURES             Location/Qualifiers
     source          1..5134
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             958..1665
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1103..1121)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1371..1390
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1710..1727)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1716..1940
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1986..2414
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2073..2092)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2283..2304
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(2623..2656)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     2660..2680
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     terminator      2910..2929
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     primer_bind     complement(2977..2993)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2977..2993)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2990..3012)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3001..3017
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3025..3031,3032..3049,3050..3055))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3070..3091
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3208..3225)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3379..3967)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3459..3478)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4138..4929,4930..4998))
                     /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     4761..4780
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4999..5103)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttggta ccgagctcgg atccactagt ccagtgtggt ggaattcatg
      961 gtgagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg
     1021 cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc
     1081 ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc
     1141 gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc
     1201 gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg
     1261 atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc
     1321 gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg
     1381 cagaagaaga ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc
     1441 ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag
     1501 gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac
     1561 atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc
     1621 gccgagggcc gccactccac cggcggcatg gacgagctgt acaaggcggc cgctcgagtc
     1681 tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     1741 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     1801 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     1861 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     1921 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta
     1981 tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     2041 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     2101 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     2161 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     2221 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     2281 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     2341 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     2401 atttaacgcg aattaattct gtggaatgga tccgacgcgc catctctagg cccgcgccgg
     2461 ccccctcgca cagacttgtg ggagaagctc ggctactccc ctgccccggt taatttgcat
     2521 ataatatttc ctagtaacta tagaggctta atgtgcgata aaagacagat aatctgttct
     2581 ttttaatact agctacattt tacatgatag gcttggattt ctataacttc gtatagcata
     2641 cattatacga agttataaac agcacaaaag gaaactcacc ctaactgtaa agtaattgtg
     2701 tgttttgaga ctataagtat cccttggaga accaccttgt tggtacgttc tctatcactg
     2761 atagttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
     2821 agtgtgtatg tgatctgata ccagcctcat agcttgagga tcacccaagt ggagcggaaa
     2881 cgctggccct tggcagcaga tcgcatactt ttttttgttt tttatgtctc atgtctgtat
     2941 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     3001 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     3061 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     3121 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     3181 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     3241 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     3301 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     3361 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     3421 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     3481 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     3541 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     3601 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     3661 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     3721 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     3781 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     3841 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     3901 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     3961 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     4021 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     4081 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     4141 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     4201 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     4261 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     4321 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     4381 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     4441 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     4501 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     4561 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     4621 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     4681 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     4741 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     4801 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     4861 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     4921 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     4981 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     5041 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     5101 gcgcacattt ccccgaaaag tgccacctga cgtc
//
