LOCUS       sequence-448203-        8895 bp DNA     circular SYN 08-OCT-2024
DEFINITION  Stable expression and inducible recruitment of protein in mammalian 
            cell culture.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8895)
  AUTHORS   Martens S, Hurley J
  TITLE     Martens and Hurley Lab Plasmids 2024
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8895)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 8, 2024 from SnapGene Server 7.0.3
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
FEATURES             Location/Qualifiers
     source          1..8895
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..634
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    681..806
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1303..1536
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1721..1765
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2062..2179
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2231..2534
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2535..2738
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2689..2709
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2735..2759
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2821..2830
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2827..3150
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     CDS             3157..3477
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /note="synthetic gene with alternative codons"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             3157..3477
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             3496..4209
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3538..3559)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3799..3818)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4146..4167
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    5285..5873
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5338..5358)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5756..5767)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5948..6181
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(6300..6318)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6386..6490
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6491..7351
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6709..6728)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7522..8110
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8011..8030
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8264..8281
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8521..8542
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        8557..8587
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8595..8611
                     /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     8600..8622
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8619..8635
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8619..8635
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     8685..8704
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
      121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
      181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
      241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
      301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
      361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
      781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
      841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
      901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
      961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa ttaattcaca
     2041 aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg
     2101 ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat
     2161 tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag tttggactag
     2221 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
     2281 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
     2341 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
     2401 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
     2461 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
     2521 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
     2581 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
     2641 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
     2701 gtaggcgtgt acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg
     2761 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccggcgg ccgcggtacc
     2821 gccaccatgg gagtgcaggt ggaaaccatc tccccaggag acgggcgcac cttccccaag
     2881 cgcggccaga cctgcgtggt gcactacacc gggatgcttg aagatggaaa gaaatttgat
     2941 tcctcccggg acagaaacaa gccctttaag tttatgctag gcaagcagga ggtgatccga
     3001 ggctgggaag aaggggttgc ccagatgagt gtgggtcaga gagccaaact gactatatct
     3061 ccagattatg cctatggtgc cactgggcac ccaggcatca tcccaccaca tgccactctc
     3121 gtcttcgatg tggagcttct aaaactggaa actagaggcg tgcaggtcga gaccatcagc
     3181 cccggcgacg gccgcacctt tcccaagaga ggccagactt gcgtggtcca ctacaccggc
     3241 atgctggagg acggcaagaa gttcgacagc agccgcgacc gcaacaagcc cttcaagttc
     3301 atgctgggca aacaggaagt gatccgcggc tgggaggaag gcgtggctca gatgagcgtg
     3361 gggcagcggg ccaagctgac catcagcccc gactatgcct acggcgccac cggccacccc
     3421 ggcatcatcc ccccccacgc caccctggtg ttcgacgtgg agctgctgaa gctggagact
     3481 agtaccggtt ctagagtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     3541 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
     3601 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     3661 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
     3721 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     3781 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     3841 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     3901 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     3961 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     4021 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     4081 cccgacaacc actacctgag cacccagtcc aagctgagca aagaccccaa cgagaagcgc
     4141 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     4201 ctgtacaagt ccggactcag atctcgagct caagcttcga attctaatct gctgccttgt
     4261 aatccacacg ggaacgggct gctgtatgct gggtttaatc aggaccacgg ctgcttcgct
     4321 tgcggaatgg agaacgggtt cagagtgtac aataccgacc cactgaaaga gaaggaaaaa
     4381 caggagttcc tggaaggcgg ggtcggacat gtggaaatgc tgtttaggtg caactacctg
     4441 gctctggtgg gaggcgggaa gaaacccaag tatcccccta acaaagtgat gatctgggac
     4501 gatctgaaga aaaagactgt gatcgagatt gagttcagca ccgaggtcaa ggccgtgaaa
     4561 ctgcggagag accgcatcgt ggtcgtgctg gattccatga tcaaggtctt cacctttaca
     4621 cacaaccctc atcagctgca cgtgttcgaa acatgctata accccaaggg cctgtgcgtg
     4681 ctgtgcccta attcaaacaa tagcctgctg gcttttcccg gaactcatac cggccacgtc
     4741 cagctggtgg acctggcatc tactgagaag ccacccgtcg atatccctgc acatgaaggg
     4801 gtgctgtcct gtatcgccct gaatctgcag ggaacacgaa ttgctactgc atctgagaag
     4861 ggcaccctga tccggatttt cgacacaagc tccggccacc tgattcagga actgaggcgc
     4921 gggagtcagg ccgctaacat ctactgcatt aacttcaatc aggacgccag cctgatctgc
     4981 gtgagcagtg atcatggcac cgtgcacatt tttgcagccg aggatccaaa gcgaaacaag
     5041 cagtcaagcc tggccagtgc ttcattcctg cccaagtatt tttcctctaa atggagcttc
     5101 tccaagtttc aggtgccttc tgggagtcca tgcatctgtg cctttggaac agagcctaac
     5161 gcagtgatcg ccatttgtgc tgatggcagc tactataaat tcctgtttaa tccaaagggg
     5221 gagtgtatta gggatgtcta tgctcagttc ctggagatga ccgacgataa gctgtaagga
     5281 tcctaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     5341 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     5401 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
     5461 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
     5521 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
     5581 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
     5641 gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct
     5701 gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
     5761 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
     5821 tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcctgagat
     5881 cctttaagac caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag
     5941 gggggactgg aagggctaat tcactcccaa cgaagacaag atctgctttt tgcttgtact
     6001 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
     6061 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
     6121 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
     6181 agtagtagtt catgtcatct tattattcag tatttataac ttgcaaagaa atgaatatca
     6241 gagagtgaga ggcccgggtt aattaaggaa agggctagat cattcttgaa gacgaaaggg
     6301 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     6361 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     6421 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     6481 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     6541 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     6601 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     6661 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     6721 ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     6781 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     6841 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     6901 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     6961 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     7021 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     7081 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     7141 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     7201 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     7261 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     7321 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     7381 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     7441 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     7501 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     7561 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     7621 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     7681 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     7741 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     7801 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     7861 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     7921 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     7981 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     8041 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     8101 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     8161 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     8221 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     8281 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     8341 atgcagcaag ctcatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     8401 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     8461 aagctccccg tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     8521 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
     8581 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgacatgat
     8641 tacgaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     8701 catcaatgta tcttatcatg tctggatcaa ctggataact caagctaacc aaaatcatcc
     8761 caaacttccc accccatacc ctattaccac tgccaattac ctgtggtttc atttactcta
     8821 aacctgtgat tcctctgaat tattttcatt ttaaagaaat tgtatttgtt aaatatgtac
     8881 tacaaactta gtagt
//
