LOCUS       sequence-445598-        7904 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transfection of USP27X (K181E) with HA tag in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7904)
  AUTHORS   Koch I, Slovik M, Zhang Y, Liu B, Rennie M, Konz E, Cogne B, Daana 
            M, Davids L, Diets IJ, Gold NB, Holtz AM, Isidor B, Mor-Shaked H, 
            Neira Fresneda J, Niederhoffer KY, Nizon M, Pfundt R, Simon M, 
            Stegmann A, Guillen Sacoto MJ, Wevers M, Barakat TS, Yanovsky-Dagan 
            S, Atanassov BS, Toth R, Gao C, Bustos F, Harel T
  TITLE     USP27X variants underlying X-linked intellectual disability disrupt 
            protein function via distinct mechanisms.
  JOURNAL   Life Sci Alliance. 2024 Jan 5;7(3):e202302258. doi: 
            10.26508/lsa.202302258. Print 2024 Mar.
  PUBMED    38182161
REFERENCE   2  (bases 1 to 7904)
  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: "Life Sci 
            Alliance."; date: "2024-01-5"; volume: "7(3):e202302258. doi"; 
            pages: " 10.26508/lsa.202302258. Print 2024 Mar"
COMMENT     Sequence Label: pCAGGS puro HA-hUSP27X K181E
FEATURES             Location/Qualifiers
     source          1..7904
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     448..467
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     701..718
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    835..856
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(871..876,877..894,895..901)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    909..925
                     /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)."
     primer_bind     914..936
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     933..949
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     933..949
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     979..998
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        979..997
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     enhancer        1007..1386
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          1682..2698
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2622..2644
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2706..2725
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2762..2788
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    4499..4633
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4536..4555)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4590..4609
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        4638..5137
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4673..4696)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     5070..5089
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             5153..5752
                     /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(5153..5172)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5649..5669
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(6088..6105)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6088..6104)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6097..6119)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(6245..6700)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6382..6403)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6594..6613
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        6727..6831
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6832..6900,6901..7692)
                     /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     complement(7050..7069)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7863..7904,1..547)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
       61 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
      121 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
      181 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
      241 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
      301 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
      361 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
      421 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      481 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      541 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      601 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
      661 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
      721 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
      781 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
      841 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
      901 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgaca tgattacgaa
      961 tttggccaag tcggcctcta atacgactca ctatagggag ctcgtcgaca ttgattattg
     1021 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
     1081 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
     1141 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
     1201 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
     1261 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
     1321 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
     1381 accatggtcg aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca
     1441 cccccaattt tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg
     1501 gggggggggg ccccaggcgg gggggggggg gggggggggg gggggggggg gggggggggg
     1561 ggggggggcg gggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
     1621 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
     1681 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
     1741 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
     1801 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
     1861 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg
     1921 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg
     1981 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg
     2041 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt
     2101 gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca
     2161 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
     2221 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     2281 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg
     2341 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
     2401 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
     2461 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
     2521 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     2581 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     2641 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     2701 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tcgccaccat
     2761 gtacccatac gatgtgccag attacgccgg atccatgtgt aaggactatg tatatgacaa
     2821 agacattgag caaattgcca aagaagagca aggagaagct ttgaaattac aagcctccac
     2881 ctcaacagag gtttctcacc agcagtgttc agtgccaggc cttggtgaga aattcccaac
     2941 ctgggaaaca accaaaccag aattagaact gctggggcac aacccgagga gaagaagaat
     3001 cacctccagc tttacgatcg gtttaagagg actcatcaat cttggcaaca cgtgctttat
     3061 gaactgcatt gtccaggccc tcacccacac gccgatactg agagatttct ttctctctga
     3121 caggcaccga tgtgagatgc cgagtcccga gttgtgtctg gtctgtgaga tgtcgtcgct
     3181 gtttcgggag ttgtattctg gaaacccgtc tcctcatgtg ccctataagt tactgcacct
     3241 ggtgtggata catgcccgcc atttagcagg gtacaggcaa caggatgccc acgagttcct
     3301 cattgcagcg ttagatgtcc tgcacaggca ctgcgaaggt gatgatgtcg ggaaggcggc
     3361 caacaatccc aaccactgta actgcatcat agaccaaatc ttcacaggtg gcctgcagtc
     3421 tgatgtcacc tgtcaagcct gccatggcgt ctccaccacg atagacccat gctgggacat
     3481 tagtttggac ttgcctggct cttgcacctc cttctggccc atgagcccag ggagggagag
     3541 cagtgtgaac ggggaaagcc acataccagg aatcaccacc ctcacggact gcttgcggag
     3601 gtttacgagg ccagagcact taggaagcag tgccaaaatc aaatgtggta gttgccaaag
     3661 ctaccaggaa tctaccaaac agctcacaat gaataaatta cctgtcgttg cctgttttca
     3721 tttcaaacgg tttgaacatt cagcgaaaca gaggcgcaag atcactacat acatttcctt
     3781 tcctctggag ctggatatga cgccgtttat ggcctcaagt aaagagagca gaatgaatgg
     3841 acaattgcag ctgccaacca atagtggaaa caacgaaaat aagtattcct tgtttgctgt
     3901 ggttaatcac caaggaacct tggagagtgg ccactatacc agcttcatcc ggcaccacaa
     3961 ggaccagtgg ttcaagtgtg atgatgccgt catcactaag gccagtatta aggacgtact
     4021 ggacagtgaa gggtatttac tgttctatca caaacaggtg ctagaacatg agtcagaaaa
     4081 agtgaaagaa atgaacacac aagcctactg aggatccggc aagcttggta cctcgcgact
     4141 cgagccttaa ttaagggcta gcaaatgcat gcggccgcca attccagctg agcgccggtc
     4201 gctaccatta ccagttggtc tggtgtcaaa aataataata accgggcagg ccatgtctgc
     4261 ccgtatttcg cgtaaggaaa tccattatgt actatttaaa aaacacaaac ttttggatgt
     4321 tcggtttatt ctttttcttt tactttttta tcatgggagc ctacttcccg tttttcccga
     4381 tttggctaca tgacatcaac catatcagca aaagtgatac gggtattatt tttgccgcta
     4441 tttctctgtt ctcgctatta ttccaaccgc tgtttggtct gctttctgac aaactcggaa
     4501 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     4561 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     4621 tcatgtctgg atctaccggg taggggaggc gcttttccca aggcagtctg gagcatgcgc
     4681 tttagcagcc ccgctgggca cttggcgcta cacaagtggc ctctggcctc gcacacattc
     4741 cacatccacc ggtaggcgcc aaccggctcc gttctttggt ggccccttcg cgccaccttc
     4801 tactcctccc ctagtcagga agttcccccc cgccccgcag ctcgcgtcgt gcaggacgtg
     4861 acaaatggaa gtagcacgtc tcactagtct cgtgcagatg gacagcaccg ctgagcaatg
     4921 gaagcgggta ggcctttggg gcagcggcca atagcagctt tgctccttcg ctttctgggc
     4981 tcagaggctg ggaaggggtg ggtccggggg cgggctcagg ggcgggctca ggggcggggc
     5041 gggcgcccga aggtcctccg gaggcccggc attctgcacg cttcaaaagc gcacgtctgc
     5101 cgcgctgttc tcctcttcct catctccggg cctttcgaca agctagctta ccatgaccga
     5161 gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct
     5221 cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat
     5281 cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa
     5341 ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga
     5401 agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct
     5461 ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg
     5521 gttcctggcc accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt
     5581 cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc
     5641 cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga
     5701 ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gacgcccgcc
     5761 ccacgacccg cagcgcccga ccgaaaggag cgcacgaccc catgcatcga tttgccagcc
     5821 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     5881 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     5941 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     6001 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctcgacgcg
     6061 gccggccgct gaccaagtca gcttggcact ggccgtcgtt ttacaacgtc gtgactggga
     6121 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     6181 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     6241 atggaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     6301 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     6361 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     6421 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     6481 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     6541 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     6601 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     6661 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac ttttcgggga
     6721 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     6781 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     6841 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     6901 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     6961 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     7021 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     7081 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     7141 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     7201 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     7261 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     7321 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     7381 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     7441 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     7501 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     7561 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     7621 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     7681 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     7741 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     7801 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     7861 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaac
//
