LOCUS       sequence-252730-        7417 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Theophylline switch Theo-CAUAA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7417)
  AUTHORS   Xiang JS, Kaplan M, Dykstra P, Hinks M, McKeague M, Smolke CD
  TITLE     Massively parallel RNA device engineering in mammalian cells with 
            RNA-Seq.
  JOURNAL   Nat Commun. 2019 Sep 23;10(1):4327. doi: 10.1038/s41467-019-12334-y.
  PUBMED    31548547
REFERENCE   2  (bases 1 to 7417)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2019-09-23"; volume: "10(1):4327. doi"; pages: " 
            10.1038/s41467-019-12334-y"
COMMENT     Sequence Label: pCS4092
FEATURES             Location/Qualifiers
     source          1..7417
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        48..259
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             272..540
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             570..1271
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     primer_bind     complement(1328..1347)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1372..1419
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     complement(1632..1651)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1823..2202
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2203..2406
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2357..2377
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2451..2470
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2451..2469
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2500..2509
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2506..3216
                     /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     2919..2938
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(3369..3386)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3375..3599
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3732..3751)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3883..3930
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3938..4960
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3938..4960
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    5090..5211
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5127..5146)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5181..5200
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5260..5276)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5260..5276)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5273..5295)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5284..5300
                     /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(5308..5314,5315..5332,5333..5338))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5353..5374
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5491..5508)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5662..6250)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5742..5761)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6421..7212,7213..7281))
                     /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     7044..7063
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7282..7386)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctgga tctgcgatcg ctccggtgcc cgtcagtggg cagagcgcac
       61 atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgaaccg gtgcctagag
      121 aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga
      181 gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg
      241 gtttgccgcc agaacacagc tgaagcttcg aggggctcgc atctctcctt cacgcgcccg
      301 ccgccctacc tgaggccgcc atccacgccg gttgagtcgc gttctgccgc ctcccgcctg
      361 tggtgcctcc tgaactgcgt ccgccgtcta ggtaagttta aagctcaggt cgagaccggg
      421 cctctgtccg gcgctccctt ggagcctacc tagactcagc cggctctcca cgctttgcct
      481 gaccctgctt gctcaactct acgtctttgt ttcgttttct gttctgcgcc gttacagatc
      541 caagctgtga ccctcgagta ttcgccacca tgagcgagct gattaaggag aacatgcaca
      601 tgaagctgta catggagggc accgtggaca accatcactt caagtgcaca tccgagggcg
      661 aaggcaagcc ctacgagggc acccagacca tgagaatcaa ggtggtcgag ggcggccctc
      721 tccccttcgc cttcgacatc ctggctacta gcttcctcta cggcagcaag accttcatca
      781 accacaccca gggcatcccc gacttcttca agcagtcctt ccctgagggc ttcacatggg
      841 agagagtcac cacatacgaa gacgggggcg tgctgaccgc tacccaggac accagcctcc
      901 aggacggctg cctcatctac aacgtcaaga tcagaggggt gaacttcaca tccaacggcc
      961 ctgtgatgca gaagaaaaca ctcggctggg aggccttcac cgagacgctg taccccgctg
     1021 acggcggcct ggaaggcaga aacgacatgg ccctgaagct cgtgggcggg agccatctga
     1081 tcgcaaacat caagaccaca tatagatcca agaaacccgc taagaacctc aagatgcctg
     1141 gcgtctacta tgtggactac agactggaaa gaatcaagga ggccaacaac gagacctacg
     1201 tcgagcagca cgaggtggca gtggccagat actgcgacct ccctagcaaa ctggggcaca
     1261 agcttaattg atctgcatga gacgatctca tgctggagtt cttcgcccac cctaggggga
     1321 ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa
     1381 aaagacagaa taaaacgcac ggtgttgggt cgtttgttca taaacgcggg gttcggtccc
     1441 agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac gcccgcgttt
     1501 cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc gcagccaacg
     1561 tcggggcggc aggccctgcc atagcctcag gttactcata gatctcccga tcccctatgg
     1621 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct
     1681 tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc
     1741 ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat
     1801 gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt
     1861 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
     1921 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
     1981 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
     2041 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
     2101 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
     2161 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
     2221 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
     2281 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
     2341 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
     2401 agagctctct ggctaactag agaacccact gcttactggc ttatcgaaat taatacgact
     2461 cactataggg agacccaagc tggctagcac gctacggtcg ccaccatggt gagcaagggc
     2521 gaggaggata acatggccat catcaaggag ttcatgcgct tcaaagtgca catggagggc
     2581 tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc
     2641 acccagaccg ccaagctgaa agtgaccaag ggtggccccc tgcccttcgc ctgggacatc
     2701 ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc
     2761 gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag
     2821 gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac
     2881 aaagtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc
     2941 atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag
     3001 atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaagt caagaccacc
     3061 tacaaggcca agaagcccgt gcagctgccc ggagcctaca acgtcaacat caagttggac
     3121 atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc cgagggccgc
     3181 cactccaccg gcggcatgga cgagctgtac aagtaactga tcataaatat agggcccgga
     3241 tccaaacaaa caaagctgtc accggaatac cagcatcgtc ttgatgccct tggaagtccg
     3301 gtctgatgag tcccataagg acgaaacagc aaaaagaaaa ataaaaaggt accaaacccg
     3361 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     3421 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     3481 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     3541 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     3601 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     3661 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     3721 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     3781 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     3841 cgaccccaaa aaacttgatt agggtgatgg ttcacgtacc tagaagttcc tattccgaag
     3901 ttcctattct ctagaaagta taggaacttc cttggccaaa aagcctgaac tcaccgcgac
     3961 gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc
     4021 ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg
     4081 ggtaaatagc tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc
     4141 ggccgcgctc ccgattccgg aagtgcttga cattggggaa ttcagcgaga gcctgaccta
     4201 ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc
     4261 cgctgttctg cagccggtcg cggaggccat ggatgcgatc gctgcggccg atcttagcca
     4321 gacgagcggg ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga
     4381 tttcatatgc gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac
     4441 cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc
     4501 cgaagtccgg cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg
     4561 ccgcataaca gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt
     4621 cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt
     4681 cgagcggagg catccggagc ttgcaggatc gccgcggctc cgggcgtata tgctccgcat
     4741 tggtcttgac caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc
     4801 gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat
     4861 cgcccgcaga agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg
     4921 aaaccgacgc cccagcactc gtccgagggc aaaggaatag cacgtactac gagatttcga
     4981 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     5041 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
     5101 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     5161 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     5221 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     5281 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     5341 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     5401 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     5461 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     5521 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     5581 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     5641 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     5701 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     5761 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     5821 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     5881 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     5941 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     6001 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     6061 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg
     6121 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     6181 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     6241 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     6301 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     6361 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     6421 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     6481 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     6541 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     6601 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     6661 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     6721 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     6781 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     6841 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     6901 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     6961 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     7021 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     7081 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     7141 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     7201 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     7261 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     7321 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     7381 tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
