LOCUS       sequence-417081-        7634 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient mammalian expression of the CC functionalized GEMS 
            receptor A-a2-GEMS (PLCG).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7634)
  AUTHORS   Makri Pistikou AM, Cremers GAO, Nathalia BL, Meuleman TJ, Bogels 
            BWA, Eijkens BV, de Dreu A, Bezembinder MTH, Stassen OMJA, Bouten 
            CCV, Merkx M, Jerala R, de Greef TFA
  TITLE     Engineering a scalable and orthogonal platform for synthetic 
            communication in mammalian cells.
  JOURNAL   Nat Commun. 2023 Nov 2;14(1):7001. doi: 10.1038/s41467-023-42810-5.
  PUBMED    37919273
REFERENCE   2  (bases 1 to 7634)
  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"; journalName: "Nat 
            Commun."; date: "2023-11-2"; volume: "14(1):7001. doi"; pages: " 
            10.1038/s41467-023-42810-5"
COMMENT     Sequence Label: pLeo-A-a2-GEMS(PLCG)
FEATURES             Location/Qualifiers
     source          1..7634
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        18..214
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      65..200
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      240..249
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             315..341
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2998..3015)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3004..3228
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3274..3702
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3361..3380)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3571..3592
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3711..3731)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3716..4045
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3896..4031
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4112..4906
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4166..4185)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4776..4795
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5080..5201
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5117..5136)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5171..5190
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5250..5266)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5250..5266)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5263..5285)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5274..5290
                     /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(5298..5304,5305..5322,5323..5328))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5343..5364
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5481..5498)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5652..6240)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5732..5751)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6411..7202,7203..7271))
                     /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     7034..7053
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7272..7376)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7451..7470)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 acgcgtgatc tgcgatctgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
       61 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatc gctgactaat
      121 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
      181 aggaggcttt tttggaggcc taggcttttg caaaaagctt cgaatcgcgc tagcggccgg
      241 ccaccatgga aactgatact ttgctgctct gggtcctgct gctgtgggtc cctggatcaa
      301 cgggggacgg atcctaccca tacgatgttc cagattacgc tggtacctcc ggccctggca
      361 gcagccccga ggacgagatc cagcaactgg aagaggaaat cgcccagctg gagcagaaaa
      421 acgccgctct gaaggaaaag aaccaggccc tgaagtacga attcggatca gggtctggaa
      481 gtgggagtgc accttcaccc agcctcccgg accccaagtt tgagagcaaa gcggccctgc
      541 tggcatcccg gggctccgaa gaacttctgt gcttcaccca acgcttggaa gacttggtgt
      601 gtttctggga ggaagcggcg agctccggga tggacttcaa ctacagcttc tcataccagc
      661 tcgagggtga gtcacgaaag tcatgtagcc tgcaccaggc tcccaccgtc cgcggctccg
      721 tgcgtttctg gtgttcactg ccaacagcgg acacatcgag tgctgtgccg ctggagctgc
      781 aggtgacgga ggcgtccggt tctcctcgct atcaccgcat catccatatc aatgaagtag
      841 tgctcctgga cgcccccgcg gggctgctgg cgcgccgggc agaagagggc agccacgtgg
      901 tgctgcgctg gctgccacct cctggagcac ctatgaccac ccacatccga tatgaagtgg
      961 acgtgtcggc aggcaaccgg gcaggaggga cacaaagggt ggaggtcctg gaaggccgca
     1021 ctgagtgtgt tctgagcaac ctgcggggcg ggacgcgcta caccttcgct gttcgagcgc
     1081 gcatggccga gccgagcttc agcggattct ggagtgcctg gtctgagccc gcgtcactac
     1141 tgaccgctag cgacctggac cctctcatct tgacgctgtc tctcattctg gtcctcatct
     1201 cgttgttgct gacggttctg gccctgctat cactacggac cgttaagcgg gccaatggag
     1261 gggaactgaa gacaggctac ttgtccatcg tcatggatcc agatgaactc ccattggatg
     1321 aacattgtga acgactgcct tatgatgcca gcaaatggga attccccaga gaccggctga
     1381 agctaggtaa gcctcttggc cgtggtgcct ttggccaagt gattgaagca gatgcctttg
     1441 gaattgacaa gacagcaact tgcaggacag tagcagtcaa aatgttgaaa gaaggagcaa
     1501 cacacagtga gcatcgagct ctcatgtctg aactcaagat cctcattcat attggtcacc
     1561 atctcaatgt ggtcaacctt ctaggtgcct gtaccaagcc aggagggcca ctcatggtga
     1621 ttgtggaatt ctgcaaattt ggaaacctgt ccacttacct gaggagcaag agaaatgaat
     1681 ttgtccccta caagaccaaa ggggcacgat tccgtcaagg gaaagactac gttggagcaa
     1741 tccctgtgga tctgaaacgg cgcttggaca gcatcaccag tagccagagc tcagccagct
     1801 ctggatttgt ggaggagaag tccctcagtg atgtagaaga agaggaagct cctgaagatc
     1861 tgtataagga cttcctgacc ttggagcatc tcatctgtta cagcttccaa gtggctaagg
     1921 gcatggagtt cttggcatcg cgaaagtgta tccacaggga cctggcggca cgaaatatcc
     1981 tcttatcgga gaagaacgtg gttaaaatct gtgactttgg cttggcccgg gatatttata
     2041 aagatccaga ttatgtcaga aaaggagatg ctcgcctccc tttgaaatgg atggccccag
     2101 aaacaatttt tgacagagtg tacacaatcc agagtgacgt ctggtctttt ggtgttttgc
     2161 tgtgggaaat attttcctta ggtgcttctc catatcctgg ggtaaagatt gatgaagaat
     2221 tttgtaggcg attgaaagaa ggaactagaa tgagggcccc tgattatact acaccagaaa
     2281 tgtaccagac catgctggac tgctggcacg gggagcccag tcagagaccc acgttttcag
     2341 agttggtgga acatttggga aatctcttgc aagctaatgc tcagcaggat ggcaaagact
     2401 acattgttct tccgatatca gagactttga gcatggaaga ggattctgga ctctctctgc
     2461 ctacctcacc tgtttcctgt atggaggagg aggaagtatg tgaccccaaa ttccattatg
     2521 acaacacagc aggaatcagt cagtatctgc agaacagtaa gcgaaagagc cggcctgtga
     2581 gtgtaaaaac atttgaagat atcccgttag aagaaccaga agtaaaagta atcccagatg
     2641 acaaccagac ggacagtggt atggttcttg cctcagaaga gctgaaaact ttggaagaca
     2701 gaaccaaatt atctccatct tttggtggaa tggtgcccag caaaagcagg gagtctgtgg
     2761 catctgaagg ctcaaaccag acaagcggct accagtccgg atatcactcc gatgacacag
     2821 acaccaccgt gtactccagt gaggaagcag aacttttaaa gctgatagag attggagtgc
     2881 aaaccggtag cacagcccag attctccagc ctgactcggg gaccacactg agctctcctc
     2941 ctgttacgcg tacgcggccg ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct
     3001 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     3061 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     3121 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     3181 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg
     3241 aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg
     3301 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     3361 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     3421 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     3481 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     3541 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     3601 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     3661 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg
     3721 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     3781 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     3841 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     3901 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     3961 ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt
     4021 ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc
     4081 tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     4141 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     4201 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     4261 gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct
     4321 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     4381 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     4441 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     4501 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     4561 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     4621 gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     4681 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     4741 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     4801 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     4861 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     4921 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     4981 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     5041 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     5101 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     5161 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     5221 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     5281 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     5341 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     5401 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     5461 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     5521 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     5581 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     5641 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     5701 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     5761 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     5821 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     5881 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     5941 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     6001 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     6061 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     6121 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     6181 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     6241 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     6301 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     6361 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     6421 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     6481 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     6541 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     6601 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     6661 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     6721 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     6781 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     6841 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     6901 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     6961 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     7021 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     7081 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     7141 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     7201 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     7261 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     7321 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     7381 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt
     7441 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
     7501 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
     7561 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
     7621 tacgggccag atat
//
