LOCUS       78X9PP_3_rph-ILV        7172 bp DNA     circular SYN 06-AUG-2024
DEFINITION  rpH-ILV-FRB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7172)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 6, 2024 from SnapGene 7.2.1
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7172
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     polyA_signal    828..962
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     906..925
                     /label=sv40pA rSeq
     promoter        complement(968..1163)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      982..1117
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     protein_bind    1222..1243
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1258..1263,1264..1281,1282..1288)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1296..1312
                     /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)."
     primer_bind     1320..1336
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    1697..1752
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1755..1791)
                     /label=pCAG-R-Seq
     CDS             complement(1911..2189)
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     CDS             complement(2196..2219)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(2325..3032)
                     /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"
     CDS             complement(2325..2342)
                     /codon_start=1
                     /label=end of XFPs
                     /translation="MDELYK"
     primer_bind     complement(2600..2619)
                     /label=mCherry F seq
                     /note="from Addgene"
     CDS             complement(3306..4019)
                     /codon_start=1
                     /product="pH-sensitive mutant of green fluorescent protein 
                     (Ng et al., 2002)"
                     /label=superecliptic pHluorin
                     /note="excited strongly by blue light at neutral pH but 
                     minimally fluorescent at acidic pH"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNDHQVYIMADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     intron          4329..5345
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     complement(4363..4401)
                     /label=pCAG-F-Seq
     promoter        complement(5346..5625)
                     /label=chicken beta-actin promoter
     enhancer        5627..6006
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6037..6141
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6142..6210,6211..7002)
                     /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"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagcggat ccagacatga
      841 taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta
      901 tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag
      961 ttagcttttt gcaaaagcct aggcctccaa aaaagcctcc tcactacttc tggaatagct
     1021 cagaggccga ggcggcctcg gcctctgcat aaataaaaaa aattagtcag ccatggggcg
     1081 gagaatgggc ggaactgggc ggagttaggg gcgggatggg cggagttagg ggcgggacta
     1141 tggttgctga ctaattgaga tgcggatccg ctggcacgac aggtttcccg actggaaagc
     1201 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
     1261 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
     1321 aggaaacagc tatgaccatg attacgccaa gcttgggctg caggtcgagg gatctccata
     1381 agagaagagg gacagctatg actgggagta gtcaggagag gaggaaaaat ctggctagta
     1441 aaacatgtaa ggaaaatttt agggatgtta aagaaaaaaa taacacaaaa caaaatataa
     1501 aaaaaatcta acctcaagtc aaggcttttc tatggaataa ggaatggaca gcagggggct
     1561 gtttcatata ctgatgacct ctttatagcc aacctttgtt catggcagcc agcatatggg
     1621 catatgttgc caaactctaa accaaatact cattctgatg ttttaaatga tttgccctcc
     1681 catatgtcct tccgagtgag agacacaaaa aattccaaca cactattgca atgaaaataa
     1741 atttccttta ttagccagaa gtcagatgct caaggggctt catgatgtcc ccataatttt
     1801 tggcagaggg aaaaagattc tcagtggtat ttgtgagcca gggcattggc cacaccagcc
     1861 accaccttct gataggcagc ctgcacctga ggagtgaatt cttagatggg ctttgagatt
     1921 cgtcggaaca catgataata gaggtcccag gcttgggtga ggtccttgac attccctgat
     1981 ttcatgtact tcctgcacca ctcttgggcc tccattaaat ctcgaccata ggcctgatta
     2041 aaggatgttt ccttcagagt ctgggggccc cgttccatca tagcatgcaa gggctccagc
     2101 acctcaaaca tgcctttcac gttcctttcc ccaaagtaca aacgagatgc ctcttccagg
     2161 ccttcatgcc acatctcatg ccagaggatc ccatcaggcc cttgaaacag aacctccaat
     2221 ctccttattt tgaccgtata tatcaacaga caaatcagtg agcatataag ttccagagca
     2281 agtattgcca gaatgaggtc cagatacagc cgtccgctag ctcccttgta cagctcgtcc
     2341 atgccgccgg tggagtggcg gccctcggcg cgttcgtact gttccacgat ggtgtagtcc
     2401 tcgttgtggg aggtgatgtc caacttgatg ttgacgttgt aggcgccggg cagctgcacg
     2461 ggcttcttgg ccttgtaggt ggtcttgacc tcagcgtcgt agtggccgcc gtccttcagc
     2521 ttcagcctct gcttgatctc gcccttcagg gcgccgtcct cggggtacat ccgctcggag
     2581 gaggcctccc agcccatggt cttcttctgc attacggggc cgtcggaggg gaagttggtg
     2641 ccgcgcagct tcaccttgta gatgaactcg ccgtcctgca gggaggagtc ctgggtcacg
     2701 gtcaccacgc cgccgtcctc gaagttcatc acgcgctccc acttgaagcc ctcggggaag
     2761 gacagcttca agtagtcggg gatgtcggcg gggtgcttca cgtaggcctt ggagccgtac
     2821 atgaactgag gggacaggat gtcccaggcg aagggcaggg ggccaccctt ggtcaccttc
     2881 agcttggcgg tctgggtgcc ctcgtagggg cggccctcgc cctcgccctc gatctcgaac
     2941 tcgtggccgt tcacggagcc ctccatgtgc accttgaagc gcatgaactc cttgatgatg
     3001 gccatgttat cctcctcgcc cttgctcacc ataccgctag caccgcctgg ttccgggaaa
     3061 gaatgttgca tacacaagat caacaagagt acagtatttc cagaggactg aatcatcaac
     3121 gcaagtctct tcagatgccg cgtggatcga tatataagat tgtatccgcg atttctgatt
     3181 tttgaatgat ggtattgtat atagcactcc aggaggagat gcagtatcca gagtataact
     3241 ttacccagaa tgatcactgt ttgcactttg agaggattgg tgtagttccc aggacatttc
     3301 ccgggtttgt atagttcatc catgccatgt gtaatcccag cagctgttac aaactcaaga
     3361 aggaccatgt ggtctctctt ttcgttggga tctttcgaaa gagtagaagt tgtaaacagg
     3421 taatggttgt ctggtaaaag gacgggccca tcgccaattg gagtattttg ttgataatgg
     3481 tctgctagtt gaacgcctcc atcttcaatg ttgtgtctaa ttttgaagtt agctttgatt
     3541 ccattctttt gtttgtctgc catgatgtac acctggtgat cgttatagtt gtattccaat
     3601 ttgtgtccaa gaatgtttcc atcttcttta aaatcaatac cttttaactc gattctatta
     3661 acaagggtat caccttcaaa cttgacttca gcacgtgtct tgtagttccc gtcatctttg
     3721 aaaaatatag ttctttcctg tacataacct tcgggcatgg cactcttgaa aaagtcatgc
     3781 cgtttcatat gatctgggta tcttgaaaag cattgaacgc cataagttaa agtagtgaca
     3841 agtgttggcc aaggaacagg tagttttcca gtagtgcaaa taaatttaag ggtaagtttt
     3901 ccgtatgttg catcaccttc accctctcca ctgacagaaa atttgtgccc attaacatca
     3961 ccatctaatt caacaagaat tgggacaact ccagtgaaaa gttcttctcc tttactcatc
     4021 ccgggcccac cgtcctcgtt ggggttaggg taactgcaaa gaacaccagt aagaaaggca
     4081 agaactacaa aaacgagatg tataaaccac aggaggttca ctatgataac tgttggcaga
     4141 ggatgaaagc gcgggcgaaa atgcgcttgc aaactatgat gtggcaaaag ctgagcgtcg
     4201 agcaaggggt cgtcctctat ggattgagta atgtcaagag aaccatcctc cattcgacct
     4261 ccagcagcca tggtgaattc tttgccaaaa tgatgagaca gcacaataac cagcacgttg
     4321 cccaggagct gtaggaaaaa gaagaaggca tgaacatggt tagcagaggc tctagagccg
     4381 ccggtcacac gccagaagcc gaaccccgcc ctgccccgtc ccccccgaag gcagccgtcc
     4441 ccccgcggac agccccgagg ctggagaggg agaaggggac ggcggcgcgg cgacgcacga
     4501 aggccctccc cgcccatttc cttcctgccg gcgccgcacc gcttcgcccc gcgcccgcta
     4561 gagggggtgc ggcggcgcct cccagatttc ggctccgcac agatttggga caaaggaagt
     4621 ccctgcgccc tctcgcacga ttaccataaa aggcaatggc tgcggctcgc cgcgcctcga
     4681 cagccgccgg cgctccgggg gccgccgcgc ccctcccccg agccctcccc ggcccgaggc
     4741 ggccccgccc cgcccggcac ccccacctgc cgccaccccc cgcccggcac ggcgagcccc
     4801 gcgccacgcc ccgtacggag ccccgcaccc gaagccgggc cgtgctcagc aactcgggga
     4861 ggggggtgca gggggggttg cagcccgacc gacgcgccca caccccctgc tcaccccccc
     4921 acgcacacac cccgcacgca gcctttgttc ccctcgcagc cccccccgca ccgcggggca
     4981 ccgcccccgg ccgcgctccc ctcgcgcaca ctgcggagcg cacaaagccc cgcgccgcgc
     5041 ccgcagcgct cacagccgcc gggcagcgcg gagccgcacg cggcgctccc cacgcacaca
     5101 cacacgcacg caccccccga gccgctcccc ccgcacaaag ggccctcccg gagcccctca
     5161 aggctttcac gcagccacag aaaagaaaca agccgtcatt aaaccaagcg ctaattacag
     5221 cccggaggag aagggccgtc ccgcccgctc acctgtggga gtaacgcggt cagtcagagc
     5281 cggggcgggc ggcgcgaggc ggcggcggag cggggcacgg ggcgaaggca gcgcgcagcg
     5341 actcccgccc gccgcgcgct tcgcttttta tagggccgcc gccgccgccg cctcgccata
     5401 aaaggaaact ttcggagcgc gccgctctga ttggctgccg ccgcacctct ccgcctcgcc
     5461 ccgccccgcc cctcgccccg ccccgccccg cctggcgcgc gccccccccc cccccccccg
     5521 cccccatcgc tgcacaaaat aattaaaaaa taaataaata caaaattggg ggtggggagg
     5581 ggggggagat ggggagagtg aagcagaacg tggggctcac ctcgaccatg gtaatagcga
     5641 tgactaatac gtagatgtac tgccaagtag gaaagtccca taaggtcatg tactgggcat
     5701 aatgccaggc gggccattta ccgtcattga cgtcaatagg gggcgtactt ggcatatgat
     5761 acacttgatg tactgccaag tgggcagttt accgtaaata ctccacccat tgacgtcaat
     5821 ggaaagtccc tattggcgtt actatgggaa catacgtcat tattgacgtc aatgggcggg
     5881 ggtcgttggg cggtcagcca ggcgggccat ttaccgtaag ttatgtaacg cggaactcca
     5941 tatatgggct atgaactaat gaccccgtaa ttgattacta ttaataacta gtcaataatc
     6001 aatgtcgacc caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     6061 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     6121 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     6181 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     6241 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     6301 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     6361 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     6421 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     6481 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     6541 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     6601 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     6661 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     6721 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     6781 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     6841 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     6901 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     6961 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     7021 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     7081 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     7141 tcagaccccg tagaaaagat caaaggatct tc
//
