LOCUS       sequence-448292-        8095 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  overexpressing N-terminus truncation of TSC22D2 with C-terminal 
            FLAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8095)
  AUTHORS   Xiao YX, Lee SY, Aguilera-Uribe M, Samson R, Au A, Khanna Y, Liu Z, 
            Cheng R, Aulakh K, Wei J, Farias AG, Reilly T, Birkadze S, Habsid A,
            Brown KR, Chan K, Mero P, Huang JQ, Billmann M, Rahman M, Myers C, 
            Andrews BJ, Youn JY, Yip CM, Rotin D, Derry WB, Forman-Kay JD, Moses
            AM, Pritisanac I, Gingras AC, Moffat J
  TITLE     The TSC22D, WNK, and NRBP gene families exhibit functional buffering
            and evolved with Metazoa for cell volume regulation.
  JOURNAL   Cell Rep. 2024 Jul 23;43(7):114417. doi: 
            10.1016/j.celrep.2024.114417. Epub 2024 Jul 8.
  PUBMED    38980795
REFERENCE   2  (bases 1 to 8095)
  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: "Cell Rep.";
            date: "2024-07-23"; volume: "43(7):114417. doi"; pages: " 
            10.1016/j.celrep.2024.114417. Epub 2024 Jul 8"
COMMENT     Sequence Label: pLJM1-TSC22D2-Nterm-C-FLAG
FEATURES             Location/Qualifiers
     source          1..8095
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    103..224
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(114..133)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     168..187
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     LTR             296..529
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     CDS             complement(657..1256)
                     /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(740..760)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     1237..1256
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(1278..1788)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1341..1359)
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     misc_feature    1837..1954
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             complement(1999..2022)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(2798..3001)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2827..2847)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     complement(3290..3310)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     CDS             complement(3608..3652)
                     /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"
     misc_feature    3837..4070
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    4563..4688
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             4735..4915
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(4916..5142)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     primer_bind     complement(5170..5190)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5170..5188)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5209..5225)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5209..5225)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5222..5244)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5233..5249
                     /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(5257..5263,5264..5281,5282..5287))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5302..5323
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5440..5457)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5611..6199)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5691..5710)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6370..7161,7162..7230))
                     /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     6993..7012
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7231..7335)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7361..7816)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7498..7519)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7710..7729
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7943..7965
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7957..7974
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7958..7974
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7984..8003
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7984..8002
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      join(8023..8095,1..63)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 aaaaaaaatt agtcagccat ggggcggaga atgggcggaa ctgggcggag ttaggggcgg
       61 gatgggcgga gttaggggcg ggatagctag agccagacat gataagatac attgatgagt
      121 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
      181 ctattgcttt atttgtaacc attataagct gcaataaaca agttcctctc actctctgat
      241 attcatttct ttgcaagtta taaatactga ataataagat gacatgaact actactgcta
      301 gagattttcc acactgacta aaagggtctg agggatctct agttaccaga gtcacacaac
      361 agacgggcac acactacttg aagcactcaa ggcaagcttt attgaggctt aagcagtggg
      421 ttccctagtt agccagagag ctcccaggct cagatctggt ctaaccagag agacccagta
      481 caagcaaaaa gcagatcttg tcttcgttgg gagtgaatta gcccttccag tccccccttt
      541 tcttttaaaa agtggctaag atctacagct gccttgtaag tcattggtct taaaggtacc
      601 gatgcatggg gtcgtgcgct cctttcggtc gggcgctgcg ggtcgtgggg cgggcgtcag
      661 gcaccgggct tgcgggtcat gcaccaggtg cgcggtcctt cgggcacctc gacgtcggcg
      721 gtgacggtga agccgagccg ctcgtagaag gggaggttgc ggggcgcgga ggtctccagg
      781 aaggcgggca ccccggcgcg ctcggccgcc tccactccgg ggagcacgac ggcgctgccc
      841 agacccttgc cctggtggtc gggcgagacg ccgacggtgg ccaggaacca cgcgggctcc
      901 ttgggccggt gcggcgccag gaggccttcc atctgttgct gcgcggccag ccgggaaccg
      961 ctcaactcgg ccatgcgcgg gccgatctcg gcgaacaccg cccccgcttc gacgctctcc
     1021 ggcgtggtcc agaccgccac cgcggcgccg tcgtccgcga cccacacctt gccgatgtcg
     1081 agcccgacgc gcgtgaggaa gagttcttgc agctcggtga cccgctcgat gtggcggtcc
     1141 ggatcgacgg tgtggcgcgt ggcggggtag tcggcgaacg cggcggcgag ggtgcgtacg
     1201 gccctgggga cgtcgtcgcg ggtggcgagg cgcaccgtgg gcttgtactc ggtcatggta
     1261 agctccggtg gatccccctg gggagagagg tcggtgattc ggtcaacgag ggagccgact
     1321 gccgacgtgc gctccggagg cttgcagaat gcggaacacc gcgcgggcag gaacagggcc
     1381 cacactaccg ccccacaccc cgcctcccgc accgcccctt cccggccgct gctctcggcg
     1441 cgccctgctg agcagccgct attggccaca gcccatcgcg gtcggcgcgc tgccattgct
     1501 ccctggcgct gtccgtctgc gagggtacta gtgagacgtg cggcttccgt ttgtcacgtc
     1561 cggcacgccg cgaaccgcaa ggaaccttcc cgacttaggg gcggagcagg aagcgtcgcc
     1621 ggggggccca caagggtagc ggcgaagatc cgggtgacgc tgcgaacgga cgtgaagaat
     1681 gtgcgagacc cagggtcggc gccgctgcgt ttcccggaac cacgcccaga gcagccgcgt
     1741 ccctgcgcaa acccagggct gccttggaaa aggcgcaacc ccaaccccct cgagccgcgg
     1801 ccaaagtgga tctctgctgt ccctgtaata aacccgaaaa ttttgaattt ttgtaatttg
     1861 tttttgtaat tctttagttt gtatgtctgt tgctattatg tctactattc tttcccctgc
     1921 actgtacccc ccaatccccc cttttctttt aaaattgtgg atgaatactg ccatttgtct
     1981 cgaggtcgag aattcttact tgtcatcgtc gtccttgtag tccacagcag tcaaggagct
     2041 gtcagttccc gactcgggcc cgtgcgcccc ctgcatggag gccactacta ccaccaccga
     2101 ccctccggtg gcgcccaggc cgctgtcccg ctccgcagtc tggtcaaaag tactgctgtg
     2161 tctaatgcaa tccccggatc tagtcaggac gctgctgtct gaatccctct catagtattc
     2221 catacacgtc catcggccgc gtctataggg ctctccgctc ccgtggtcca gcttgatcac
     2281 gcgaaaacgg gaactacatg tggtgggagg ctgagatggg gctgcagtca ctggcccggc
     2341 ggatgagccc gcgagctggg ggccgccggg tgctccgggg gcgggggccg aagtggcagc
     2401 cgccgccagg gtactggcga gcgccccaga cacgctccgg gccgaaacga ctcctcctcc
     2461 gttggcggga gcagcagccg ccgctgccag ctgcccatct aggaggaggt ttggggagac
     2521 ggtcccggga gtctccgcat ccccaacatt gttaagcgtc tcttcggaag agctgcgctc
     2581 gcagacctcc tcagggccat aatccgtggc ccgagagacg tcgaaaatct cggaagagac
     2641 gtcctctgtg cgtgactcgt ccgggtcgtc caagctctcg gtgtcctcgg tgatgctagt
     2701 ggccacctgg gccgtggtga cactggtgat ctggaagcag ctcttcttct tggccggcat
     2761 cttggacatg ctagcggatc tgacggttca ctaaaccagc tctgcttata tagacctccc
     2821 accgtacacg cctaccgccc atttgcgtca atggggcgga gttgttacga cattttggaa
     2881 agtcccgttg attttggtgc caaaacaaac tcccattgac gtcaatgggg tggagacttg
     2941 gaaatccccg tgagtcaaac cgctatccac gcccattgat gtactgccaa aaccgcatca
     3001 ccatggtaat agcgatgact aatacgtaga tgtactgcca agtaggaaag tcccataagg
     3061 tcatgtactg ggcataatgc caggcgggcc atttaccgtc attgacgtca atagggggcg
     3121 tacttggcat atgatagtcc attttaaaac ataattttaa aactgcaaac tacccaagaa
     3181 attattactt tctacgtcac gtattttgta ctaatatctt tgtgtttaca gtcaaattaa
     3241 ttccaattat ctctctaaca gccttgtatc gtatatgcaa atatgaagga atcatgggaa
     3301 ataggccctc ggtgaagggg gcggccgctc gaggctagtc tcgtgatcga taccgtcgag
     3361 atccgttcac taatcgaatg gatctgtctc tgtctctctc tccaccttct tcttctattc
     3421 cttcgggcct gtcgggtccc ctcggggttg ggaggtgggt ctgaaacgat aatggtgaat
     3481 atccctgcct aactctattc actatagaaa gtacagcaaa aactattctt aaacctacca
     3541 agcctcctac tatcattatg aataatttta tataccacag ccaatttgtt atgttaaacc
     3601 aattccacaa acttgcccat ttatctaatt ccaataattc ttgttcattc ttttcttgct
     3661 ggttttgcga ttcttcaatt aaggagtgta ttaagcttgt gtaattgtta atttctctgt
     3721 cccactccat ccaggtcgtg tgattccaaa tctgttccag agatttatta ctccaactag
     3781 cattccaagg cacagcagtg gtgcaaatga gttttccaga gcaaccccaa atccccagga
     3841 gctgttgatc ctttaggtat ctttccacag ccaggattct tgcctggagc tgcttgatgc
     3901 cccagactgt gagttgcaac agatgctgtt gcgcctcaat agccctcagc aaattgttct
     3961 gctgctgcac tataccagac aataattgtc tggcctgtac cgtcagcgtc attgacgctg
     4021 cgcccatagt gcttcctgct gctcccaaga acccaaggaa caaagctcct attcccactg
     4081 ctcttttttc tctctgcacc actcttctct ttgccttggt gggtgctact cctaatggtt
     4141 caatttttac tactttatat ttatataatt cacttctcca attgtccctc atatctcctc
     4201 ctccaggtct gaagatcagc ggccgcttgc tgtgcggtgg tcttactttt gttttgctct
     4261 tcctctatct tgtctaaagc ttccttggtg tcttttatct ctatcctttg atgcacacaa
     4321 tagagggttg ctactgtatt atataatgat ctaagttctt ctgatcctgt ctgaagggat
     4381 ggttgtagct gtcccagtat ttgtctacag ccttctgatg tttctaacag gccaggatta
     4441 actgcgaatc gttctagctc cctgcttgcc catactatat gttttaattt atattttttc
     4501 tttccccctg gccttaaccg aattttttcc catcgcgatc taattctccc ccgcttaata
     4561 ctgacgctct cgcacccatc tctctccttc tagcctccgc tagtcaaaat ttttggcgta
     4621 ctcaccagtc gccgcccctc gcctcttgcc gtgcgcgctt cagcaagccg agtcctgcgt
     4681 cgagagagct cctctggttt ccctttcgct ttcaagtccc tgttcgggcg ccactgctag
     4741 agattttcca cactgactaa aagggtctga gggatctcta gttaccagag tcacacaaca
     4801 gacgggcaca cactacttga agcactcaag gcaagcttta ttgaggctta agcagtgggt
     4861 tccctagtta gccagagagc tcccaggctc agatctggtc taaccagaga gacccgttta
     4921 tgtatcgagc taggcactta aatacaatat ctctgcaatg cggcaattca gtggttcgtc
     4981 caatccatgt cagacccgtc tgttgccttc ctaataaggc acgatcgtac caccttactt
     5041 ccaccaatcg gcatgcacgg tgctttttct ctccttgtaa ggcatgttgc taactcatcg
     5101 ttaccatgtt gcaagactac aagagtattg cataagacta cattaagctt gcagctccag
     5161 cttttgttcc ctttagtgag ggttaattgc gcgcttggcg taatcatggt catagctgtt
     5221 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     5281 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     5341 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     5401 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     5461 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     5521 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     5581 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     5641 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     5701 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5761 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     5821 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     5881 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     5941 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     6001 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     6061 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     6121 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     6181 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     6241 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6301 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     6361 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     6421 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     6481 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     6541 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     6601 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     6661 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     6721 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     6781 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     6841 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     6901 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     6961 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     7021 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     7081 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     7141 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     7201 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     7261 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     7321 aataggggtt ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatatt
     7381 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     7441 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     7501 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     7561 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     7621 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     7681 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     7741 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     7801 ccgctacagg gcgcgtccca ttcgccattc aggctgcgca actgttggga agggcgatcg
     7861 gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta
     7921 agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgagcgc
     7981 gcgtaatacg actcactata gggcgaattg ggtacgtccc taggcctcca aaaaagcctc
     8041 ctcactactt ctggaatagc tcagaggccg aggcggcctc ggcctctgca taaat
//
