LOCUS       sequence-489487-        4861 bp    DNA     circular SYN 16-OCT-2025
DEFINITION  Constitutive expression of human mature TNF (with the human IgK 
            light chain secretion sequence).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4861)
  AUTHORS   Edelstein HI, Cosio A, Ezekiel ML, Corcoran WK, Morris AH, Leonard 
            JN
  TITLE     Conversion of natural cytokine receptors into orthogonal synthetic 
            biosensors.
  JOURNAL   Nat Chem Biol. 2025 Aug 22:10.1038/s41589-025-01986-1. doi: 
            10.1038/s41589-025-01986-1.
  PUBMED    40846994
REFERENCE   2  (bases 1 to 4861)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 16, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat Chem 
            Biol. 2025 Aug 22:10.1038/s41589-025-01986-1. doi: 
            10.1038/s41589-025-01986-1."
COMMENT     Sequence Label: L4275 hIgKLSS_hTNF in pcDNA backbone
FEATURES             Location/Qualifiers
     source          1..4861
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1491..1508)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1497..1721
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1767..2195
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1854..1873)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2064..2085
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2218..2414
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2265..2400
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2327..2346
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    2534..2655
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2571..2590)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2625..2644
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2704..2720)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2704..2720)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2717..2739)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2728..2744
                     /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(2752..2758,2759..2776,2777..2782))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2797..2818
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2935..2952)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3106..3694)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3186..3205)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3865..4656,4657..4725))
                     /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     4488..4507
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4726..4830)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagtcccaa gctggctagc
      901 gccaccatgg acatgagagt gcccgcccag ctgctgggcc tgctgctgct gtggctgaga
      961 ggcgccagat gcgtcagatc atcttctcga accccgagtg acaagcctgt agcccatgtt
     1021 gtagcaaacc ctcaagctga ggggcagctc cagtggctga accgccgggc caatgccctc
     1081 ctggccaatg gcgtggagct gagagataac cagctggtgg tgccatcaga gggcctgtac
     1141 ctcatctact cccaggtcct cttcaagggc caaggctgcc cctccaccca tgtgctcctc
     1201 acccacacca tcagccgcat cgccgtctcc taccagacca aggtcaacct cctctctgcc
     1261 atcaagagcc cctgccagag ggagacccca gagggggctg aggccaagcc ctggtatgag
     1321 cccatctatc tgggaggggt cttccagctg gagaagggtg accgactcag cgctgagatc
     1381 aatcggcccg actatctcga ctttgccgag tctgggcagg tctactttgg gatcattgcc
     1441 ctgtgagcgg ccgctcgagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt
     1501 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     1561 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     1621 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     1681 agtcaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     1741 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     1801 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     1861 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     1921 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     1981 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     2041 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     2101 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     2161 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
     2221 ggctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     2281 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     2341 ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     2401 taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggaggt tgggcttcgg
     2461 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     2521 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     2581 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     2641 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc
     2701 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     2761 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     2821 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     2881 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     2941 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     3001 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     3061 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     3121 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     3181 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     3241 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     3301 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     3361 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     3421 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     3481 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     3541 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     3601 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     3661 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     3721 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     3781 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     3841 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     3901 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     3961 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagatc cacgctcacc
     4021 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     4081 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     4141 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     4201 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     4261 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     4321 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     4381 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     4441 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     4501 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     4561 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     4621 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     4681 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     4741 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     4801 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     4861 c
//
