LOCUS       sequence-417082-        6756 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient mammalian expression of the CC functionalized GEMS 
            receptor B-a2-GEMS (JAK-STAT).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6756)
  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 6756)
  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-B-a2-GEMS(JAK-STAT)
FEATURES             Location/Qualifiers
     source          1..6756
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(49..68)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     659..678
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    963..1084
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1000..1019)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1054..1073
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1133..1149)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1133..1149)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1146..1168)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1157..1173
                     /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(1181..1187,1188..1205,1206..1211))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1226..1247
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1364..1381)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1535..2123)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1615..1634)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2294..3085,3086..3154))
                     /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     2917..2936
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3155..3259)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3334..3353)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3535..3731
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3582..3717
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      3757..3766
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3832..3858
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(5637..5654)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5643..5867
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5913..6341
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6000..6019)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6210..6231
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6350..6370)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6355..6684
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6535..6670
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(6751..6756,1..789)
                     /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"
ORIGIN
        1 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
       61 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
      121 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac
      181 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
      241 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
      301 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
      361 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
      421 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
      481 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag
      541 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
      601 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
      661 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
      721 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
      781 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
      841 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
      901 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
      961 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     1021 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     1081 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     1141 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     1201 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     1261 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     1321 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     1381 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     1441 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     1501 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     1561 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     1621 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     1681 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     1741 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     1801 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     1861 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     1921 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     1981 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     2041 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     2101 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     2161 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     2221 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     2281 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     2341 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     2401 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     2461 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     2521 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     2581 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     2641 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     2701 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     2761 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     2821 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     2881 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     2941 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     3001 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     3061 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     3121 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     3181 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     3241 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg
     3301 gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     3361 aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat
     3421 ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag
     3481 gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgtgatctgc gatctgcatc
     3541 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     3601 ccagttccgc ccattctccg ccccatcgct gactaatttt ttttatttat gcagaggccg
     3661 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     3721 gcttttgcaa aaagcttcga atcgcgctag cggccggcca ccatggaaac tgatactttg
     3781 ctgctctggg tcctgctgct gtgggtccct ggatcaacgg gggacggatc ctacccatac
     3841 gatgttccag attacgctgg tacctccggc cctggcagca gccccgagga cgagaacgcc
     3901 gctctggaag agaagatcgc ccagctgaaa cagaagaacg ccgccctgaa ggaagagatc
     3961 caggccctgg aatacgaatt cggatcaggg tctggaagtg ggagtgcacc ttcacccagc
     4021 ctcccggacc ccaagtttga gagcaaagcg gccctgctgg catcccgggg ctccgaagaa
     4081 cttctgtgct tcacccaacg cttggaagac ttggtgtgtt tctgggagga agcggcgagc
     4141 tccgggatgg acttcaacta cagcttctca taccagctcg agggtgagtc acgaaagtca
     4201 tgtagcctgc accaggctcc caccgtccgc ggctccgtgc gtttctggtg ttcactgcca
     4261 acagcggaca catcgagtgc tgtgccgctg gagctgcagg tgacggaggc gtccggttct
     4321 cctcgctatc accgcatcat ccatatcaat gaagtagtgc tcctggacgc ccccgcgggg
     4381 ctgctggcgc gccgggcaga agagggcagc cacgtggtgc tgcgctggct gccacctcct
     4441 ggagcaccta tgaccaccca catccgatat gaagtggacg tgtcggcagg caaccgggca
     4501 ggagggacac aaagggtgga ggtcctggaa ggccgcactg agtgtgttct gagcaacctg
     4561 cggggcggga cgcgctacac cttcgctgtt cgagcgcgca tggccgagcc gagcttcagc
     4621 ggattctgga gtgcctggtc tgagcccgcg tcactactga ccgctagcga cctggaccct
     4681 ctcatcttga cgctgtctct cattctggtc ctcatctcgt tgttgctgac ggttctggcc
     4741 ctgctgtccg cagcagcaaa taagcgagac ctaattaaaa aacacatctg gcctaatgtt
     4801 ccagatcctt caaagagtca tattgcccag tggtcacctc acactcctcc aaggcacaat
     4861 tttaattcaa aagatcaaat gtattcagat ggcaatttca ctgatgtaag tgttgtggaa
     4921 atagaagcaa atgacaaaaa gccttttcca gaagatctga aatcattgga cctgttcaaa
     4981 aaggaaaaaa ttaatactga aggacacagc agtggtattg gggggtcttc atgcatgtca
     5041 tcttctaggc caagcatttc tagcagtgat gaaaatgaat cttcacaaaa cacttcgagc
     5101 actgtccagg cttctaccgt ggtacacagt ggctacagac accaagttcc gtcagtccaa
     5161 gtcttctcaa gatccgagtc tacccagccc ttgttagatt cagaggagcg gccagaagat
     5221 ctacaattag tagatcatgt agatggcggt gatggtattt tgcccaggca acagtacttc
     5281 aaacagaact gcagtcagca tgaatccagt ccagatattt cacattttga aaggtcaaag
     5341 caagtttcat cagtcaatga ggaagatttt gttagactta aacagcagat ttcagatcat
     5401 atttcacaat cctgtggatc tgggcaaatg aaaatgtttc aggaagtttc tgcagcagat
     5461 gcttttggtc caggtactga gggacaagta gaaagatttg aaacagttgg catggaggct
     5521 gcgactgatg aaggcatgcc taaaagttac ttaccacaga ctgtacggca aggcggctac
     5581 atgcctcagt aggcggccgc tcgagtctag agggcccgtt taaacccgct gatcagcctc
     5641 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     5701 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     5761 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     5821 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     5881 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     5941 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     6001 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     6061 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     6121 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     6181 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     6241 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     6301 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     6361 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     6421 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     6481 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     6541 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     6601 tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt
     6661 tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct
     6721 gatcaagaga caggatgagg atcgtttcgc atgatt
//
