LOCUS       sequence-448375-        7690 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  overexpressing C-terminus truncation of NRBP1 with N-terminal FLAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7690)
  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 7690)
  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-NRBP1-Cterm-N-FLAG
FEATURES             Location/Qualifiers
     source          1..7690
                     /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(2338..2361)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(2393..2596)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2422..2442)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     complement(2885..2905)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     CDS             complement(3203..3247)
                     /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    3432..3665
                     /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    4158..4283
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             4330..4510
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(4511..4737)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     primer_bind     complement(4765..4785)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4765..4783)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4804..4820)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4804..4820)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4817..4839)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4828..4844
                     /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(4852..4858,4859..4876,4877..4882))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4897..4918
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5035..5052)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5206..5794)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5286..5305)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5965..6756,6757..6825))
                     /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     6588..6607
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6826..6930)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6956..7411)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7093..7114)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7305..7324
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7538..7560
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7552..7569
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7553..7569
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7579..7598
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7579..7597
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      join(7618..7690,1..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 aattcctaag aggagacggt gacagcggct gagttgaggg tactgttcct
     2041 ggcaaaattg aacttgttca aggtctcttc tagcagagaa gtcaaccggc tctggtcagc
     2101 ctcactaatg aagcccagct gcaccagctc agccgccaac tcggggatat tctcatttgg
     2161 catcaggtca cagctcaggt gccggttcag tttgtcctcc aacttcagca gaagtgtcag
     2221 gtggtgtttg actccctcct ccaccgactc aatgttgcac tgcatcagca ccaccttgcg
     2281 agtctccacc tcagctggtt caggtgtcgg agtcttgaca gaggggggca cgacaggctt
     2341 gtcatcgtcg tccttgtagt ccatgctagc ggatctgacg gttcactaaa ccagctctgc
     2401 ttatatagac ctcccaccgt acacgcctac cgcccatttg cgtcaatggg gcggagttgt
     2461 tacgacattt tggaaagtcc cgttgatttt ggtgccaaaa caaactccca ttgacgtcaa
     2521 tggggtggag acttggaaat ccccgtgagt caaaccgcta tccacgccca ttgatgtact
     2581 gccaaaaccg catcaccatg gtaatagcga tgactaatac gtagatgtac tgccaagtag
     2641 gaaagtccca taaggtcatg tactgggcat aatgccaggc gggccattta ccgtcattga
     2701 cgtcaatagg gggcgtactt ggcatatgat agtccatttt aaaacataat tttaaaactg
     2761 caaactaccc aagaaattat tactttctac gtcacgtatt ttgtactaat atctttgtgt
     2821 ttacagtcaa attaattcca attatctctc taacagcctt gtatcgtata tgcaaatatg
     2881 aaggaatcat gggaaatagg ccctcggtga agggggcggc cgctcgaggc tagtctcgtg
     2941 atcgataccg tcgagatccg ttcactaatc gaatggatct gtctctgtct ctctctccac
     3001 cttcttcttc tattccttcg ggcctgtcgg gtcccctcgg ggttgggagg tgggtctgaa
     3061 acgataatgg tgaatatccc tgcctaactc tattcactat agaaagtaca gcaaaaacta
     3121 ttcttaaacc taccaagcct cctactatca ttatgaataa ttttatatac cacagccaat
     3181 ttgttatgtt aaaccaattc cacaaacttg cccatttatc taattccaat aattcttgtt
     3241 cattcttttc ttgctggttt tgcgattctt caattaagga gtgtattaag cttgtgtaat
     3301 tgttaatttc tctgtcccac tccatccagg tcgtgtgatt ccaaatctgt tccagagatt
     3361 tattactcca actagcattc caaggcacag cagtggtgca aatgagtttt ccagagcaac
     3421 cccaaatccc caggagctgt tgatccttta ggtatctttc cacagccagg attcttgcct
     3481 ggagctgctt gatgccccag actgtgagtt gcaacagatg ctgttgcgcc tcaatagccc
     3541 tcagcaaatt gttctgctgc tgcactatac cagacaataa ttgtctggcc tgtaccgtca
     3601 gcgtcattga cgctgcgccc atagtgcttc ctgctgctcc caagaaccca aggaacaaag
     3661 ctcctattcc cactgctctt ttttctctct gcaccactct tctctttgcc ttggtgggtg
     3721 ctactcctaa tggttcaatt tttactactt tatatttata taattcactt ctccaattgt
     3781 ccctcatatc tcctcctcca ggtctgaaga tcagcggccg cttgctgtgc ggtggtctta
     3841 cttttgtttt gctcttcctc tatcttgtct aaagcttcct tggtgtcttt tatctctatc
     3901 ctttgatgca cacaatagag ggttgctact gtattatata atgatctaag ttcttctgat
     3961 cctgtctgaa gggatggttg tagctgtccc agtatttgtc tacagccttc tgatgtttct
     4021 aacaggccag gattaactgc gaatcgttct agctccctgc ttgcccatac tatatgtttt
     4081 aatttatatt ttttctttcc ccctggcctt aaccgaattt tttcccatcg cgatctaatt
     4141 ctcccccgct taatactgac gctctcgcac ccatctctct ccttctagcc tccgctagtc
     4201 aaaatttttg gcgtactcac cagtcgccgc ccctcgcctc ttgccgtgcg cgcttcagca
     4261 agccgagtcc tgcgtcgaga gagctcctct ggtttccctt tcgctttcaa gtccctgttc
     4321 gggcgccact gctagagatt ttccacactg actaaaaggg tctgagggat ctctagttac
     4381 cagagtcaca caacagacgg gcacacacta cttgaagcac tcaaggcaag ctttattgag
     4441 gcttaagcag tgggttccct agttagccag agagctccca ggctcagatc tggtctaacc
     4501 agagagaccc gtttatgtat cgagctaggc acttaaatac aatatctctg caatgcggca
     4561 attcagtggt tcgtccaatc catgtcagac ccgtctgttg ccttcctaat aaggcacgat
     4621 cgtaccacct tacttccacc aatcggcatg cacggtgctt tttctctcct tgtaaggcat
     4681 gttgctaact catcgttacc atgttgcaag actacaagag tattgcataa gactacatta
     4741 agcttgcagc tccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc
     4801 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     4861 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     4921 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4981 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     5041 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     5101 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     5161 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     5221 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     5281 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     5341 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     5401 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     5461 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5521 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     5581 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5641 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     5701 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     5761 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     5821 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     5881 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     5941 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     6001 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     6061 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     6121 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     6181 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     6241 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     6301 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     6361 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     6421 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     6481 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     6541 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     6601 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     6661 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     6721 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     6781 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     6841 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     6901 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt
     6961 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     7021 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     7081 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     7141 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     7201 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     7261 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     7321 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     7381 gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct gcgcaactgt
     7441 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
     7501 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
     7561 acggccagtg agcgcgcgta atacgactca ctatagggcg aattgggtac gtccctaggc
     7621 ctccaaaaaa gcctcctcac tacttctgga atagctcaga ggccgaggcg gcctcggcct
     7681 ctgcataaat
//
