LOCUS       sequence-448376-        5164 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  endogenously tag TSC22D2 with C-terminal mScarlet.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5164)
  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 5164)
  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: pYX-C-mScarlet-Zeo-TSC22D2
FEATURES             Location/Qualifiers
     source          1..5164
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             963..1655
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="VSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGGA
                     VTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKM
                     ALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHS
                     TGGMDELYK"
     primer_bind     complement(1093..1111)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1361..1380
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1662..1679
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1689..1745
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1752..2123
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="AKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDDV
                     TLFISAVQDQVVPDNTLAWVLVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGRE
                     FALRDPAGNCVHFVAEEQD"
     polyA_signal    2130..2354
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2943..2959)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2943..2959)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2956..2978)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2967..2983
                     /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(2991..2997,2998..3015,3016..3021))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3036..3057
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3174..3191)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3345..3933)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3425..3444)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4104..4895,4896..4964))
                     /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     4727..4746
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4965..5069)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5137..5155
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cttttttttt aattctaagc
      421 ttagttgaca atttaagaag ctagtatttt ttggaaatcg tcccaaactt tgaaaagaaa
      481 attctgggct tactgctctg gttgggaact ggattttttc caacccatct tccataggct
      541 cggtcaagat aaagcagaaa cattaaaagc aagagcagga aaactagtat aaaaacttag
      601 caccaagtag gccagaacca gatagcacca cctttatctt ttcactcttt tgacattcct
      661 aattttgttt ttcacaggat ctggtgaaaa gccatttgat gtatgcagta agagaagaag
      721 tggaagtttt aaaggaacaa ataaaagaat tagttgaaag aaactcttta cttgaacgag
      781 aaaatgcact gttaaaatct ctttcaagca atgatcaatt atcccaactc ccaacccaac
      841 aggccaatcc tggtagcact tctcaacagc aagcagtgat agcacagcct ccgcagccaa
      901 cgcaacctcc acaacaaccg aatgtctcct cagcaggagg aagcggagga tctggcggta
      961 ccgtgagcaa gggcgaggca gtgatcaagg agttcatgcg gttcaaggtg cacatggagg
     1021 gctccatgaa cggccacgag ttcgagatcg agggcgaggg cgagggacga ccctacgagg
     1081 gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc tcctgggaca
     1141 tcctgtcccc tcagttcatg tacggctcca gggccttcat caagcacccc gccgacatcc
     1201 ccgactacta taagcagtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg
     1261 aggacggcgg cgccgtgacc gtgacccagg acacctccct ggaggacggc accctgatct
     1321 acaaggtgaa gctccgcggc accaacttcc ctcctgacgg ccccgtaatg cagaagaaga
     1381 caatgggctg ggaagcgtcc accgagcggt tgtaccccga ggacggcgtg ctgaagggcg
     1441 acattaagat ggccctgcgc ctgaaggacg gcggacgcta cctggcggac ttcaagacca
     1501 cctacaaggc caagaagccc gtgcagatgc ccggcgccta caacgtcgac cgcaagttgg
     1561 acatcacctc ccacaacgag gactacaccg tggtggaaca gtacgaacgc tccgagggcc
     1621 gccactccac cggaggcatg gacgagctgt ataagctgca gcaccatcac caccatcacg
     1681 gatccggagc tactaacttc agcctgctga agcaggccgg agacgtggag gagaaccctg
     1741 gaccttctag agccaagttg accagtgccg ttccggtgct caccgcgcgc gacgtcgccg
     1801 gagcggtcga gttctggacc gaccggctcg ggttctcccg ggacttcgtg gaggacgact
     1861 tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag cgcggtccag gaccaggtgg
     1921 tgccggacaa caccctggcc tgggtgctgg tgcgcggcct ggacgagctg tacgccgagt
     1981 ggtcggaggt cgtgtccacg aacttccggg acgcctccgg gccggccatg accgagatcg
     2041 gcgagcagcc gtgggggagg gagttcgccc tgcgcgatcc ggccggcaac tgcgtgcact
     2101 tcgtggccga ggagcaggac taactcgagc tgtgccttct agttgccagc catctgttgt
     2161 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     2221 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     2281 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     2341 ggtgggctct atgggcggcc gctaagagca agaagtaaag ctttcttaag cctcattaag
     2401 aaaaaaactg aaagcaatct atccttgtgt gccactggtg ttctttccac tttatacgaa
     2461 agcaagtagc catgctttgg ttgtgtgttt ggccttttca gtattagaca atcattctac
     2521 aagagctttt cctctctctg agatgtcatg cagcgctgtt gatgtccagt tctatgtcat
     2581 cagtacacaa ggagaataat agatggggtt tattaaagcg agcaaagtct gcattttacc
     2641 tggtgcgcat gagtggggtc tttaagagtt ttggtggctc tcccatgttt cctattaccc
     2701 atggatttac cctgagcctt cctatcacat tataaataac agttcatcta aagagccact
     2761 tttctttctg attcagtaac atttgcctac ataagttttc atttatttgt gttttattta
     2821 ttacagggct gctattttca taatgtacat gaacaatgtc acagaacttt tttaattttt
     2881 ttgaataatt ataagtatca gtaaaggaag tgaaagacag gattaagctt ggcgtaatca
     2941 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     3001 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     3061 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     3121 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     3181 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     3241 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     3301 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     3361 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     3421 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     3481 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     3541 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     3601 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     3661 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     3721 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     3781 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     3841 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     3901 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     3961 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     4021 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     4081 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     4141 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     4201 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     4261 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     4321 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     4381 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     4441 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     4501 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     4561 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     4621 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     4681 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     4741 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     4801 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     4861 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     4921 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     4981 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     5041 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     5101 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     5161 cgtc
//
