LOCUS       sequence-386938-        8167 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses receiver cell components of LOTIIS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8167)
  AUTHORS   Ng KK, Prescher JA
  TITLE     Generalized Bioluminescent Platform To Observe and Track Cellular 
            Interactions.
  JOURNAL   Bioconjug Chem. 2022 Oct 19;33(10):1876-1884. doi: 
            10.1021/acs.bioconjchem.2c00348. Epub 2022 Sep 27.
  PUBMED    36166258
REFERENCE   2  (bases 1 to 8167)
  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"; doi: 
            "10.1021/acs.bioconjchem.2c00348"; journalName: "Bioconjug Chem"; 
            date: "2022-10-19- 19"; volume: "33"; issue: "10"; pages: 
            "1876-1884"
COMMENT     Sequence Label: 2x GCN4-LgBiT
FEATURES             Location/Qualifiers
     source          1..8167
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        163..366
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     317..337
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     411..430
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        411..429
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             540..596
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             612..668
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             684..707
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1395..1451
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1992..2048
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2049..2744
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     CDS             2772..2825
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             2826..3425
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2826..2845)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3322..3342
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3585..3706
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3622..3641)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3676..3695
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    3832..4361
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     primer_bind     complement(4711..4727)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4711..4727)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4724..4746)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4735..4751
                     /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(4759..4765,4766..4783,4784..4789))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4804..4825
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4942..4959)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5113..5701)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5193..5212)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5872..6663,6664..6732))
                     /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     6495..6514
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6733..6837)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6912..6931)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     misc_feature    7201..7702
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     enhancer        join(7950..8167,1..162)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgtcccc tattgacgtc
       61 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
      121 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
      181 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
      241 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
      301 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
      361 agagctctct ggctaactag agaacccact gcttactggc ttatcgaaat taatacgact
      421 cactataggg agacccaagc tggctagcgc taccggtcgc cacctctaga atggccttac
      481 cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg ccggaattcg
      541 aagaactttt gagcaagaat tatcatcttg agaacgaagt ggctcgtctt aagaaaggtt
      601 ctggcagtgg agaagaactg ctttcaaaga attaccacct ggaaaatgag gtagctagac
      661 tgaaaaaggg cggcggaggc tctgactaca aggacgatga cgataagggc ggaggcgggt
      721 ctttccagaa ggcctccagc atagtctata agaaagaggg ggaacaggtg gagttctcct
      781 tcccactcgc ctttacagtt gaaaagctga cgggcagtgg cgagctgtgg tggcaggcgg
      841 agagggcttc ctcctccaag tcttggatca cctttgacct gaagaacaag gaagtgtctg
      901 taaaacgggt tacccaggac cctaagctcc agatgggcaa gaagctcccg ctccacctca
      961 ccctgcccca ggccttgcct cagtatgctg gctctggaaa cctcaccctg gcccttgaag
     1021 cgaaaacagg aaagttgcat caggaagtga acctggtggt gatgagagcc actcagctcc
     1081 agaaaaattt gacctgtgag gtgtggggac ccacctcccc taagctgatg ctgagcttga
     1141 aactggagaa caaggaggca aaggtctcga agcgggagaa ggcggtgtgg gtgctgaacc
     1201 ctgaggcggg gatgtggcag tgtctgctga gtgactcggg acaggtcctg ctggaatcca
     1261 acatcaaggt tctgcccaca tggtccaccc cggtgcagcc aatggccctg attgtgctgg
     1321 ggggcgtcgc cggcctcctg cttttcattg ggctaggcat cttcttctgt gtcaggtgcc
     1381 ggcaccgaag gcgcgctact aacttcagcc tgctgaagca ggctggagac gtggaggaga
     1441 accctggacc tatgtggtgg cgcctgtggt ggctgctgct gctgctgctg ctgctgtggc
     1501 ccatggtgtg ggccatcgat ttcacactcg aagatttcgt tggggactgg gaacagacag
     1561 ccgcctacaa cctggaccaa gtccttgaac agggaggtgt gtccagtttg ctgcagaatc
     1621 tcgccgtgtc cgtaactccg atccaaagga ttgtccggag cggtgaaaat gccctgaaga
     1681 tcgacatcca tgtcatcatc ccgtatgaag gtctgagcgc cgaccaaatg gcccagatcg
     1741 aagaggtgtt taaggtggtg taccctgtgg atgatcatca ctttaaggtg atcctgccct
     1801 atggcacact ggtaatcgac ggggttacgc cgaacatgct gaactatttc ggacggccgt
     1861 atgaaggcat cgccgtgttc gacggcaaaa agatcactgt aacagggacc ctgtggaacg
     1921 gcaacaaaat tatcgacgag cgcctgatca cccccgacgg ctccatgctg ttccgagtaa
     1981 ccatcaacag cgccacgaac ttctctctgt taaagcaagc aggagacgtg gaagaaaacc
     2041 ccggtcctag cgagctgatt aaggagaaca tgcacatgaa gctgtacatg gagggcaccg
     2101 tggacaacca tcacttcaag tgcacatccg agggcgaagg caagccctac gagggcaccc
     2161 agaccatgag aatcaaggtg gtcgagggcg gccctctccc cttcgccttc gacatcctgg
     2221 ctactagctt cctctacggc agcaagacct tcatcaacca cacccagggc atccccgact
     2281 tcttcaagca gtccttccct gagggcttca catgggagag agtcaccaca tacgaagacg
     2341 ggggcgtgct gaccgctacc caggacacca gcctccagga cggctgcctc atctacaacg
     2401 tcaagatcag aggggtgaac ttcacatcca acggccctgt gatgcagaag aaaacactcg
     2461 gctgggaggc cttcaccgag acgctgtacc ccgctgacgg cggcctggaa ggcagaaacg
     2521 acatggccct gaagctcgtg ggcgggagcc atctgatcgc aaacatcaag accacatata
     2581 gatccaagaa acccgctaag aacctcaaga tgcctggcgt ctactatgtg gactacagac
     2641 tggaaagaat caaggaggcc aacaacgaga cctacgtcga gcagcacgag gtggcagtgg
     2701 ccagatactg cgacctccct agcaaactgg ggcacaagct taatctcgag ggcagcggcg
     2761 agggcagcgg cgagggcaga ggcagcctgc tgacctgcgg cgacgtggag gagaaccccg
     2821 gccccatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtcccccggg
     2881 ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgacc
     2941 cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc
     3001 tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc
     3061 cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga
     3121 gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca
     3181 aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc
     3241 tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct
     3301 tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca
     3361 ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg
     3421 cctgattcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     3481 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     3541 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     3601 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     3661 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatat
     3721 tgggtctaac ccccacctcc tgttaggcag attccttatc tggtgacaca cccccatttc
     3781 ctggagccat ctctctcctt gccagaacct ctaaggtttg cttacgatgg agccagagag
     3841 gatcctggga gggagagctt ggcagggggt gggagggaag ggggggatgc gtgacctgcc
     3901 cggttctcag tggccaccct gcgctaccct ctcccagaac ctgagctgct ctgacgcggc
     3961 tgtctggtgc gtttcactga tcctggtgct gcagcttcct tacacttccc aagaggagaa
     4021 gcagtttgga aaaacaaaat cagaataagt tggtcctgag ttctaacttt ggctcttcac
     4081 ctttctagtc cccaatttat attgttcctc cgtgcgtcag ttttacctgt gagataaggc
     4141 cagtagccag ccccgtcctg gcagggctgt ggtgaggagg ggggtgtccg tgtggaaaac
     4201 tccctttgtg agaatggtgc gtcctaggtg ttcaccaggt cgtggccgcc tctactccct
     4261 ttctctttct ccatccttct ttccttaaag agtccccagt gctatctggg acatattcct
     4321 ccgcccagag cagggtcccg cttccctaag gccctgctct gggcttctgg gtttgagtcc
     4381 ttggcaagcc caggagaggc gctcaggctt ccctgtcccc cttcctcgtc caccatctca
     4441 tgcccctggc tctcctgccc cttccctaca ggggttcctg gctctgctct tcagactgag
     4501 ccccgttccc ctgcatcccc gttcccctgc atcccccttc ccctgcatcc cccagaggcc
     4561 ccaggccacc tacttggcct ggaccccacg agaggccacc ccagccctgt ctaccaggct
     4621 gccttttggg tggattctcc tccaactgtg gggtgactgc ttggcaaacg aattcccgtc
     4681 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     4741 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     4801 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     4861 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     4921 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     4981 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     5041 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     5101 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     5161 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     5221 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     5281 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     5341 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     5401 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     5461 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     5521 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     5581 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     5641 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     5701 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     5761 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     5821 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     5881 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     5941 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     6001 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     6061 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     6121 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     6181 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     6241 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     6301 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     6361 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     6421 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     6481 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     6541 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     6601 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     6661 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     6721 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     6781 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     6841 atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg
     6901 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct
     6961 tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc
     7021 ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat
     7081 gtacgggcca gatatacgcg ttggtgagtg aattcctgcc gcctccttca ggttccgtct
     7141 tcctccactc cctcttcccc ttgctctctg ctgtgttgct gcccaaggat gctctttccg
     7201 gagcacttcc ttctcggcgc tgcaccacgt gatgtcctct gagcggatcc tccccgtgtc
     7261 tgggtcctct ccgggcatct ctcctccctc acccaacccc atgccgtctt cactcgctgg
     7321 gttccctttt ccttctcctt ctggggcctg tgccatctct cgtttcttag gatggccttc
     7381 tccgacggat gtctcccttg cgtcccgcct ccccttcttg taggcctgca tcatcaccgt
     7441 ttttctggac aaccccaaag taccccgtct ccctggcttt agccacctct ccatcctctt
     7501 gctttctttg cctggacacc ccgttctcct gtggattcgg gtcacctctc actcctttca
     7561 tttgggcagc tcccctaccc cccttacctc tctagtctgt gctagctctt ccagccccct
     7621 gtcatggcat cttccagggg tccgagagct cagctagtct tcttcctcca acccgggccc
     7681 ctatgtccac ttcaggacag catgtttgct gcctccaggg atcctgtgtc cccgagctgg
     7741 gaccacctta tattcccagg gccggttaat gtggctctgg ttctgggtac ttttatctgt
     7801 cccctccacc ccacagtggg gccactaggg acaggattgg tgacagaaaa gccccatcct
     7861 taggcgcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
     7921 tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta
     7981 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
     8041 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
     8101 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
     8161 acggtaa
//
