LOCUS       sequence-471115-        7041 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Protein overexpression of eGFP-human uroporphyrinogen decarboxylase 
            N367A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7041)
  TITLE     A method for the detection and enrichment of endogenous cereblon 
            substrates
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7041)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 7, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pET28a-His6-GFP-UROD-N367A
FEATURES             Location/Qualifiers
     source          1..7041
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(994..1704,1705..1707))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             complement(join(994..1704,1705..1707))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1036..1057)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1385..1404
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1644..1665
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(1708..1725)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1735..1757
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1772..1796
                     /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     complement(1796..1815)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1797..1815)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1899..1918)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2124..2201
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2202..3284
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(2221..2240)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4093..4284
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4278..4300)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4386..4525
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4441..4460
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4540..4557)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4711..5299)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4791..4810)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5410..5429)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5421..6236
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             5421..6236
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(5494..5513)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6328..6783)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6465..6486)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6677..6696
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(6820..6867)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     6863..6881
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
ORIGIN
        1 atgtggacca aagtcgtcca acatttgttt gacaagttga ccgatttcct cttcgctagc
       61 atataaagca caggggtcca gattcccctg caatgtaacc gtcttcccca cgcactcacg
      121 agctttcttc ggggctaccg tccagtcaag cccaactact tcgtagcctg cctgtgccaa
      181 ttcctccaag gcgaagtgcc catccttggc aaagatgatc ataggtactg gtgcaagacc
      241 cgcctcgcgc aaacgggcct taacttgctt ggccacatca cggatatagg gcagagcaaa
      301 tttgttaaac agctgtggtc caaggtgacc tgcgtgtgac tcaaataact gcaaggcctg
      361 cgcacctgcg acgacctgac ccacaagata aggcaccaag gcatcggtca gaatgcgtaa
      421 caattggtgc gacgcttgtg gacgttggta caaccagcgt tttgcctggg ccatagtaga
      481 gctgcctcct ccctctacca tgtaggtcat aagagtccat ggtgctccag caaatccaat
      541 cagaggaaca cgtcccgcaa gacgctggcg tgtaagggta atagcctgga agacataccc
      601 cagctcggac gccaccacct ccgggtcacg caagcgttcc aagtcctgct cttcgcgcag
      661 tggttctgga aaggacggcc ccttccctgg aaccatcgtt acctccatac ccagagcttg
      721 agggactacc aaaatgtcgc tgaagatgat cgccgcatca agtggaaaac ggcgtaacgg
      781 ctgaagagtc aattcgcagc acgcctcggg agaacggcag gtgctaaaaa aatcttgcgc
      841 cgcacgcgtt tcgcggaact ctggaaggta acggccagcc tggcgcatac accacacggg
      901 tgtatagtcc gtttcttccc cccaggctgc acgaaggaag gtatcgtttt tcaactctgg
      961 aaacccttga gggccaaggc cgttcgcttc catcttgtac agctcgtcca tgccgagagt
     1021 gatcccggcg gcggtcacga actccagcag gaccatgtga tcgcgcttct cgttggggtc
     1081 tttgctcagg gcggactggg tgctcaggta gtggttgtcg ggcagcagca cggggccgtc
     1141 gccgatgggg gtgttctgct ggtagtggtc ggcgagctgc acgctgccgt cctcgatgtt
     1201 gtggcggatc ttgaagttca ccttgatgcc gttcttctgc ttgtcggcca tgatatagac
     1261 gttgtggctg ttgtagttgt actccagctt gtgccccagg atgttgccgt cctccttgaa
     1321 gtcgatgccc ttcagctcga tgcggttcac cagggtgtcg ccctcgaact tcacctcggc
     1381 gcgggtcttg tagttgccgt cgtccttgaa gaagatggtg cgctcctgga cgtagccttc
     1441 gggcatggcg gacttgaaga agtcgtgctg cttcatgtgg tcggggtagc ggctgaagca
     1501 ctgcacgccg taggtcaggg tggtcacgag ggtgggccag ggcacgggca gcttgccggt
     1561 ggtgcagatg aacttcaggg tcagcttgcc gtaggtggca tcgccctcgc cctcgccgga
     1621 cacgctgaac ttgtggccgt ttacgtcgcc gtccagctcg accaggatgg gcaccacccc
     1681 ggtgaacagc tcctcgccct tgctcacgtg gtggtgatga tgatgcatgg tatatctcct
     1741 tcttaaagtt aaacaaaatt atttctagag gggaattgtt atccgctcac aattccccta
     1801 tagtgagtcg tattaatttc gcgggatcga gatctcgatc ctctacgccg gacgcatcgt
     1861 ggccggcatc accggcgcca caggtgcggt tgctggcgcc tatatcgccg acatcaccga
     1921 tggggaagat cgggctcgcc acttcgggct catgagcgct tgtttcggcg tgggtatggt
     1981 ggcaggcccc gtggccgggg gactgttggg cgccatctcc ttgcatgcac cattccttgc
     2041 ggcggcggtg ctcaacggcc tcaacctact actgggctgc ttcctaatgc aggagtcgca
     2101 taagggagag cgtcgagatc ccggacacca tcgaatggcg caaaaccttt cgcggtatgg
     2161 catgatagcg cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat
     2221 acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg
     2281 ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt
     2341 acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg
     2401 ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg
     2461 ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct
     2521 gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc
     2581 cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat
     2641 ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat gaagacggta
     2701 cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg
     2761 gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc
     2821 gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc
     2881 aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg
     2941 atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg
     3001 atatctcggt agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa
     3061 ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac
     3121 tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa
     3181 aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     3241 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     3301 gtaagttagc tcactcatta ggcaccggga tctcgaccga tgcccttgag agccttcaac
     3361 ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtc
     3421 ttctttatca tgcaactcgt aggacaggtg ccggcagcgc tctgggtcat tttcggcgag
     3481 gaccgctttc gctggagcgc gacgatgatc ggcctgtcgc ttgcggtatt cggaatcttg
     3541 cacgccctcg ctcaagcctt cgtcactggt cccgccacca aacgtttcgg cgagaagcag
     3601 gccattatcg ccggcatggc ggccccacgg gtgcgcatga tcgtgctcct gtcgttgagg
     3661 acccggctag gctggcgggg ttgccttact ggttagcaga atgaatcacc gatacgcgag
     3721 cgaacgtgaa gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc
     3781 ttcggtttcc gtgtttcgta aagtctggaa acgcggaagt cagcgccctg caccattatg
     3841 ttccggatct gcatcgcagg atgctgctgg ctaccctgtg gaacacctac atctgtatta
     3901 acgaagcgct ggcattgacc ctgagtgatt tttctctggt cccgccgcat ccataccgcc
     3961 agttgtttac cctcacaacg ttccagtaac cgggcatgtt catcatcagt aacccgtatc
     4021 gtgagcatcc tctctcgttt catcggtatc attaccccca tgaacagaaa tcccccttac
     4081 acggaggcat cagtgaccaa acaggaaaaa accgccctta acatggcccg ctttatcaga
     4141 agccagacat taacgcttct ggagaaactc aacgagctgg acgcggatga acaggcagac
     4201 atctgtgaat cgcttcacga ccacgctgat gagctttacc gcagctgcct cgcgcgtttc
     4261 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     4321 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     4381 cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg
     4441 cggcatcaga gcagattgta ctgagagtgc accattgcgg tgtgaaatac cgcacagatg
     4501 cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg
     4561 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     4621 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     4681 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     4741 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     4801 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     4861 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     4921 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     4981 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     5041 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     5101 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt
     5161 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     5221 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     5281 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     5341 gaacgaaaac tcacgttaag ggattttggt catgaacaat aaaactgtct gcttacataa
     5401 acagtaatac aaggggtgtt atgagccata ttcaacggga aacgtcttgc tctaggccgc
     5461 gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg
     5521 ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc cgatgcgcca gagttgtttc
     5581 tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact
     5641 ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg
     5701 catggttact caccactgcg atccccggga aaacagcatt ccaggtatta gaagaatatc
     5761 ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga
     5821 ttcctgtttg taattgtcct tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat
     5881 cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc
     5941 ctgttgaaca agtctggaaa gaaatgcata aacttttgcc attctcaccg gattcagtcg
     6001 tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt
     6061 gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga
     6121 actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg
     6181 ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaagaat
     6241 taattcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     6301 cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt taatattttg ttaaaattcg
     6361 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     6421 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
     6481 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     6541 atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     6601 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacggtga aagccggcga
     6661 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     6721 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     6781 cgtcccattc gccaatccgg atatagttcc tcctttcagc aaaaaacccc tcaagacccg
     6841 tttagaggcc ccaaggggtt atgctagtta ttgctcagcg gtggcagcag ccaactcagc
     6901 ttcctttcgg gctttgttag cagccggatc tcaagcttgg cgcagtaaac gggagtgctt
     6961 atggacggca tcaacgaatg cgcctacgtg ctctggatcc atatccgggt acagtccatg
     7021 gccaagattt gcgatatagc g
//
