LOCUS       sequence-393647-        5530 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of human GID4 with N-terminal NanoLuciferase 
            fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5530)
  AUTHORS   Owens DGD, Maitland E.R, Yazdi AK, Song X, Schwalm MP, Machado RAC, 
            Bauer N , Wang X, Szewczyk MM, Dong C , Dong A, Loppnau P, Calabrese
            MF, Dowling MS, Lee J, Montgomery JI, O'Connell TN, Subramanyam C, 
            Wang F, Schapira M, Knapp S , Vedadi M, Min J, Lajoie GA, 
            Barsyte-Lovejoy D , Owen DR, Schild-Poulter C, Arrowsmith CH
  TITLE     A chemical probe to modulate human GID4 Pro/N-degron interactions
  JOURNAL   bioRxiv 2023.01.17.524225
REFERENCE   2  (bases 1 to 5530)
  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: "bioRxiv 
            2023.01.17.524225"
COMMENT     Sequence Label: NLuc-GID4
FEATURES             Location/Qualifiers
     source          1..5530
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..721
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     672..692
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          857..989
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     972..991
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     1033..1052
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1033..1051
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1065..1577
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     primer_bind     complement(2609..2627)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    2664..2785
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2675..2694)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2729..2748
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3030..3387
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3030..3221
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3053..3073)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3238..3373
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        3402..3449
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3481..4275
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3535..3554)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4145..4164
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4339..4387
                     /note="synthetic polyadenylation signal"
     rep_origin      4544..5089
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     4990..5009
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5206..5223
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      661 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      721 tggtttagtg aaccgtcaga tcactagaag ctttattgcg gtagtttatc acagttaaat
      781 tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttaagctgca gaagttggtc
      841 gtgaggcact gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa
      901 actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac
      961 tgacatccac tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta
     1021 aggctagagt attaatacga ctcactatag ggctagcgct caccatggtc ttcacactcg
     1081 aagatttcgt tggggactgg cgacagacag ccggctacaa cctggaccaa gtccttgaac
     1141 agggaggtgt gtccagtttg tttcagaatc tcggggtgtc cgtaactccg atccaaagga
     1201 ttgtcctgag cggtgaaaat gggctgaaga tcgacatcca tgtcatcatc ccgtatgaag
     1261 gtctgagcgg cgaccaaatg ggccagatcg aaaaaatttt taaggtggtg taccctgtgg
     1321 atgatcatca ctttaaggtg atcctgcact atggcacact ggtaatcgac ggggttacgc
     1381 cgaacatgat cgactatttc ggacggccgt atgaaggcat cgccgtgttc gacggcaaaa
     1441 agatcactgt aacagggacc ctgtggaacg gcaacaaaat tatcgacgag cgcctgatca
     1501 accccgacgg ctccctgctg ttccgagtaa ccatcaacgg agtgaccggc tggcggctgt
     1561 gcgaacgcat tctggcgggc tcgagcggcg cgatcgccat gtgtgcgcga gggcaagtcg
     1621 ggagggggac ccagctcagg actgggaggc cctgctcgca ggtccctggg tcccggtggc
     1681 ggccggagcg cttgctccgc aggcagcggg cgggtggtcg cccctcccgc ccccaccccg
     1741 cgcgtgcgcg ccccggcctc tccctccccg ccaccctcct cggctcccgc gcggcggcgg
     1801 cggttcctct cccactcccc ccagccctgg ctccggggga ccccgcgatg ccggtccgca
     1861 ccgagtgtcc cccgccggcc ggtgcctccg ctgcctccgc ggcctcactc atcccgccgc
     1921 cgcccatcaa cacccagcag cccggcgtgg ccaccagcct gctctacagc ggctccaagt
     1981 tccgcggcca ccagaagagc aaggggaact cgtacgacgt agaggtggtg ctgcagcacg
     2041 tggacacggg gaactcttac ctttgtgggt acttgaagat taaaggcctt actgaggagt
     2101 atccaaccct tacaaccttc ttcgaaggag aaataatcag caaaaaacac cctttcttaa
     2161 ctcgcaagtg ggatgcagat gaagatgttg atcggaaaca ctggggcaag tttctggctt
     2221 tttatcagta tgcaaaatca tttaactcag atgactttga ttatgaagag ctgaagaatg
     2281 gagactacgt cttcatgagg tggaaggaac agtttctggt cccagatcac acgatcaaag
     2341 acatcagtgg tgcttctttt gccgggttct actacatctg ctttcagaag tcagcagcct
     2401 ccatagaggg ctactactac cataggagtt cagaatggta tcagtccctc aatctaaccc
     2461 atgttcctga acacagtgca cccatctatg aattccggta aacgaattcg ggctcggtac
     2521 ccggggatcc tctagagtcg acctgcaggc atgcaagctg atccggctgc taacaaagcc
     2581 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
     2641 gcggccgctt cgagcagaca tgataagata cattgatgag tttggacaaa ccacaactag
     2701 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     2761 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
     2821 tcagggggag atgtgggagg tttttttaag caagtaaaac ctctacaaat gtggtaaaat
     2881 cgaattttaa caaaatatta acgcttacaa tttcctgatg cggtattttc tccttacgca
     2941 tctgtgcggt atttcacacc gcatacgcgg atctgcgcag caccatggcc tgaaataacc
     3001 tctgaaagag gaacttggtt aggtaccttc tgaggcggaa agaaccagct gtggaatgtg
     3061 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3121 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     3181 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     3241 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     3301 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
     3361 ttttttggag gcctaggctt ttgcaaaaag cttaattaac tgttgacaat taatcatcgg
     3421 catagtatat cggcatagta taatacgaca aggtgaggaa ctaaacccag gaggcagatc
     3481 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     3541 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     3601 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     3661 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     3721 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     3781 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     3841 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     3901 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     3961 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     4021 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     4081 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     4141 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     4201 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     4261 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     4321 tgccatcacg atggccgcaa taaaatatct ttattttcat tacatctgtg tgttggtttt
     4381 ttgtgtgaat cgatagcgat aaggatccgc tcatgcgatg ataagctgtc aaacatgaga
     4441 attacaactt atatcgtatg gggctgactt caggtgctac atttgaagag ataaattgca
     4501 ctgaaatcta gaaatatttt atctgattaa taagatgatc ttcttgagat cgttttggtc
     4561 tgcgcgtaat ctcttgctct gaaaacgaaa aaaccgcctt gcagggcggt ttttcgaagg
     4621 ttctctgagc taccaactct ttgaaccgag gtaactggct tggaggagcg cagtcaccaa
     4681 aacttgtcct ttcagtttag ccttaaccgg cgcatgactt caagactaac tcctctaaat
     4741 caattaccag tggctgctgc cagtggtgct tttgcatgtc tttccgggtt ggactcaaga
     4801 cgatagttac cggataaggc gcagcggtcg gactgaacgg ggggttcgtg catacagtcc
     4861 agcttggagc gaactgccta cccggaactg agtgtcaggc gtggaatgag acaaacgcgg
     4921 ccataacagc ggaatgacac cggtaaaccg aaaggcagga acaggagagc gcacgaggga
     4981 gccgccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc accactgatt
     5041 tgagcgtcag atttcgtgat gcttgtcagg ggggcggagc ctatggaaaa acggctttgc
     5101 cgcggccctc tcacttccct gttaagtatc ttcctggcat cttccaggaa atctccgccc
     5161 cgttcgtaag ccatttccgc tcgccgcagt cgaacgaccg agcgtagcga gtcagtgagc
     5221 gaggaagcgg aatatatcct gtatcacata ttctgctgac gcaccggtgc agcctttttt
     5281 ctcctgccac atgaagcact tcactgacac cctcatcagt gccaacatag taagccagta
     5341 tacactccgc tagcgctgat gtccggcggt gcttttgccg ttacgcacca ccccgtcatg
     5401 agattacgcg ttttagcggt gcgtacaatt aagggattat ggtaaatcca cttactgtct
     5461 gccctcgtag ccatcgagat aaaccgcagt actccggcca cgatgcgtcc ggcgtagagg
     5521 atcgagatct
//
