LOCUS       sequence-380741-        7281 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses FRB protein in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7281)
  TITLE     Jun Liu Lab Plasmids 2022
REFERENCE   2  (bases 1 to 7281)
  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"
COMMENT     Sequence Label: pLenti6M FRB
FEATURES             Location/Qualifiers
     source          1..7281
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        42..345
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        346..549
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     500..520
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     546..570
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      632..641
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             671..940
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS
                     FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ"
     CDS             1017..1058
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(1095..1115)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1100..1429
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1280..1415
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        1477..1524
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             1543..1941
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     LTR             2028..2261
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2333..2454
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2370..2389)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2424..2443
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2494..2629
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2649..2668)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2650..2668)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2678..2695)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2678..2694)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2687..2709)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2836..3291
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2923..2942)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3133..3154
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3317..3421
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3422..3490,3491..4282)
                     /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     complement(3640..3659)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4453..5041
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4942..4961
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5195..5212
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5329..5350
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5365..5370,5371..5388,5389..5395)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5403..5419
                     /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     5408..5430
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5427..5443
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5427..5443
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5462..5482
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5464..5482
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5510..5736
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5737..5917
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5964..6089
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6582..6815
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7000..7044
                     /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"
ORIGIN
        1 gtgaacggat ctcgacggta tcgataagct tgggagttcc gcgttacata acttacggta
       61 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
      121 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
      181 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
      241 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
      301 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
      361 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
      421 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
      481 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
      541 agcagagctc gtttagtgaa ccgtcagatc gcctggagac gccatccacg ctgttttgac
      601 ctccatagaa gacaccgact ctagaggatc cgccaccatg gagctgattc gagtagccat
      661 cctctggcat gagatgtggc atgaaggcct agaagaggcc tctcgcttgt actttgggga
      721 gaggaacgtc aaaggcatgt ttgaggtgct ggagcccctg catgctatga tggaacgcgg
      781 tccccagacc ctgaaggaaa cgtcctttaa tcaggcatat ggtcgagatt taatggaggc
      841 acaagaatgg tgccgaaagt acatgaaatc agggaacgtc aaggacctca cccaagcctg
      901 ggacctctac tatcacgtgt tcagacggat ctccaagcag ggtaaaaaga agaaaaagaa
      961 gtcaaagaca aagtgtgtaa ttatgtaact cgagtctaga gggcccgcgg ttcgaaggta
     1021 agcctatccc taaccctctc ctcggtctcg attctacgcg taccggttag taatgagttt
     1081 ggaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
     1141 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
     1201 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     1261 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     1321 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct
     1381 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg
     1441 agcttgtata tccattttcg gatctgatca gcacgtgttg acaattaatc atcggcatag
     1501 tatatcggca tagtataata cgacaaggtg aggaactaaa ccatggccaa gcctttgtct
     1561 caagaagaat ccaccctcat tgaaagagca acggctacaa tcaacagcat ccccatctct
     1621 gaagactaca gcgtcgccag cgcagctctc tctagcgacg gccgcatctt cactggtgtc
     1681 aatgtatatc attttactgg gggaccttgt gcagaactcg tggtgctggg cactgctgct
     1741 gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa caggggcatc
     1801 ttgagcccct gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg gatcaaagcc
     1861 atagtgaagg acagtgatgg acagccgacg gcagttggga ttcgtgaatt gctgccctct
     1921 ggttatgtgt gggagggcta agcacaattc gagctcggta cctttaagac caatgactta
     1981 caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat
     2041 tcactcccaa cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca
     2101 gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag
     2161 cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag
     2221 atccctcaga cccttttagt cagtgtggaa aatctctagc agtagtagtt catgtcatct
     2281 tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt
     2341 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     2401 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     2461 ctggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     2521 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     2581 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct agggacgtac
     2641 ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc
     2701 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     2761 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     2821 tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     2881 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     2941 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     3001 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     3061 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     3121 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     3181 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     3241 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     3301 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     3361 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     3421 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     3481 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     3541 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     3601 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     3661 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     3721 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     3781 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     3841 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     3901 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     3961 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     4021 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     4081 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     4141 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     4201 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     4261 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     4321 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     4381 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     4441 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     4501 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     4561 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     4621 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     4681 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     4741 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     4801 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     4861 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     4921 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     4981 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     5041 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     5101 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     5161 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     5221 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     5281 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     5341 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     5401 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg
     5461 cgcaattaac cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat
     5521 gcaatactct tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga
     5581 gagaaaaagc accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta
     5641 ggaaggcaac agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga
     5701 gatattgtat ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc
     5761 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
     5821 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
     5881 ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga
     5941 aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca
     6001 cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct
     6061 agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat
     6121 gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat
     6181 gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag
     6241 gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta
     6301 gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag
     6361 acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac
     6421 agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt
     6481 gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca
     6541 aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg
     6601 ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc
     6661 agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg
     6721 caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg
     6781 gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa
     6841 ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag
     6901 atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta
     6961 atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg
     7021 gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat
     7081 ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta
     7141 ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc
     7201 ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac
     7261 agagacagat ccattcgatt a
//
