LOCUS       sequence-469915-        7041 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Protein overexpression of eGFP-human uroporphyrinogen decarboxylase.
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  JOURNAL   Unpublished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            "Unpublished"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
FEATURES             Location/Qualifiers
     source          1..7041
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(13..32)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             24..839
                     /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             24..839
                     /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(97..116)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(931..1386)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1068..1089)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1280..1299
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(1423..1470)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     1466..1484
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(join(2638..3348,3349..3351))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             complement(join(2638..3348,3349..3351))
                     /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(2680..2701)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3029..3048
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3288..3309
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(3352..3369)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             3379..3401
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    3416..3440
                     /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(3440..3459)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3441..3459)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3543..3562)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3768..3845
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3846..4928
                     /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(3865..3884)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5737..5928
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5922..5944)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    6030..6169
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6085..6104
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6184..6201)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6355..6943)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6435..6454)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtct tgctctaggc
       61 cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg
      121 tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg ccagagttgt
      181 ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa
      241 actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg
      301 atgcatggtt actcaccact gcgatccccg ggaaaacagc attccaggta ttagaagaat
      361 atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt
      421 cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc
      481 aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct
      541 ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag
      601 tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag
      661 gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat
      721 ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta
      781 ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaag
      841 aattaattca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
      901 ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat
      961 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     1021 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     1081 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     1141 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     1201 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg tgaaagccgg
     1261 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     1321 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     1381 gcgcgtccca ttcgccaatc cggatatagt tcctcctttc agcaaaaaac ccctcaagac
     1441 ccgtttagag gccccaaggg gttatgctag ttattgctca gcggtggcag cagccaactc
     1501 agcttccttt cgggctttgt tagcagccgg atctcaattt tggcgcagta aacgggagtg
     1561 cttatggacg gcatcaacga atgcgcctac gtgctctgga tccatatccg ggtacagtcc
     1621 atggccaaga tttgcgatat agcgatgtgg accaaagtcg tccaacattt gtttgacaag
     1681 ttgaccgatt tcctcttcgc tagcatataa agcacagggg tccagattcc cctgcaatgt
     1741 aaccgtcttc cccacgcact cacgagcttt cttcggggct accgtccagt caagcccaac
     1801 tacttcgtag cctgcctgtg ccaattcctc caaggcgaag tgcccatcct tggcaaagat
     1861 gatcataggt actggtgcaa gacccgcctc gcgcaaacgg gccttaactt gcttggccac
     1921 atcacggata tagggcagag caaatttgtt aaacagctgt ggtccaaggt gacctgcgtg
     1981 tgactcaaat aactgcaagg cctgcgcacc tgcgacgacc tgacccacaa gataaggcac
     2041 caaggcatcg gtcagaatgc gtaacaattg gtgcgacgct tgtggacgtt ggtacaacca
     2101 gcgttttgcc tgggccatag tagagctgcc tcctccctct accatgtagg tcataagagt
     2161 ccatggtgct ccagcaaatc caatcagagg aacacgtccc gcaagacgct ggcgtgtaag
     2221 ggtaatagcc tggaagacat accccagctc ggacgccacc acctccgggt cacgcaagcg
     2281 ttccaagtcc tgctcttcgc gcagtggttc tggaaaggac ggccccttcc ctggaaccat
     2341 cgttacctcc atacccagag cttgagggac taccaaaatg tcgctgaaga tgatcgccgc
     2401 atcaagtgga aaacggcgta acggctgaag agtcaattcg cagcacgcct cgggagaacg
     2461 gcaggtgcta aaaaaatctt gcgccgcacg cgtttcgcgg aactctggaa ggtaacggcc
     2521 agcctggcgc atacaccaca cgggtgtata gtccgtttct tccccccagg ctgcacgaag
     2581 gaaggtatcg tttttcaact ctggaaaccc ttgagggcca aggccgttcg cttccatctt
     2641 gtacagctcg tccatgccga gagtgatccc ggcggcggtc acgaactcca gcaggaccat
     2701 gtgatcgcgc ttctcgttgg ggtctttgct cagggcggac tgggtgctca ggtagtggtt
     2761 gtcgggcagc agcacggggc cgtcgccgat gggggtgttc tgctggtagt ggtcggcgag
     2821 ctgcacgctg ccgtcctcga tgttgtggcg gatcttgaag ttcaccttga tgccgttctt
     2881 ctgcttgtcg gccatgatat agacgttgtg gctgttgtag ttgtactcca gcttgtgccc
     2941 caggatgttg ccgtcctcct tgaagtcgat gcccttcagc tcgatgcggt tcaccagggt
     3001 gtcgccctcg aacttcacct cggcgcgggt cttgtagttg ccgtcgtcct tgaagaagat
     3061 ggtgcgctcc tggacgtagc cttcgggcat ggcggacttg aagaagtcgt gctgcttcat
     3121 gtggtcgggg tagcggctga agcactgcac gccgtaggtc agggtggtca cgagggtggg
     3181 ccagggcacg ggcagcttgc cggtggtgca gatgaacttc agggtcagct tgccgtaggt
     3241 ggcatcgccc tcgccctcgc cggacacgct gaacttgtgg ccgtttacgt cgccgtccag
     3301 ctcgaccagg atgggcacca ccccggtgaa cagctcctcg cccttgctca cgtggtggtg
     3361 atgatgatgc atggtatatc tccttcttaa agttaaacaa aattatttct agaggggaat
     3421 tgttatccgc tcacaattcc cctatagtga gtcgtattaa tttcgcggga tcgagatctc
     3481 gatcctctac gccggacgca tcgtggccgg catcaccggc gccacaggtg cggttgctgg
     3541 cgcctatatc gccgacatca ccgatgggga agatcgggct cgccacttcg ggctcatgag
     3601 cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc gggggactgt tgggcgccat
     3661 ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc tactactggg
     3721 ctgcttccta atgcaggagt cgcataaggg agagcgtcga gatcccggac accatcgaat
     3781 ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg
     3841 tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga
     3901 ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg
     3961 aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca
     4021 aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa
     4081 ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg
     4141 tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg
     4201 tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg
     4261 cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta
     4321 ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc
     4381 accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg
     4441 ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg
     4501 actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc
     4561 ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg
     4621 agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca
     4681 gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa
     4741 ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt
     4801 tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc
     4861 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     4921 ggcagtgagc gcaacgcaat taatgtaagt tagctcactc attaggcacc gggatctcga
     4981 ccgatgccct tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact
     5041 atcgtcgccg cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca
     5101 gcgctctggg tcattttcgg cgaggaccgc tttcgctgga gcgcgacgat gatcggcctg
     5161 tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc
     5221 accaaacgtt tcggcgagaa gcaggccatt atcgccggca tggcggcccc acgggtgcgc
     5281 atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc ggggttgcct tactggttag
     5341 cagaatgaat caccgatacg cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg
     5401 acctgagcaa caacatgaat ggtcttcggt ttccgtgttt cgtaaagtct ggaaacgcgg
     5461 aagtcagcgc cctgcaccat tatgttccgg atctgcatcg caggatgctg ctggctaccc
     5521 tgtggaacac ctacatctgt attaacgaag cgctggcatt gaccctgagt gatttttctc
     5581 tggtcccgcc gcatccatac cgccagttgt ttaccctcac aacgttccag taaccgggca
     5641 tgttcatcat cagtaacccg tatcgtgagc atcctctctc gtttcatcgg tatcattacc
     5701 cccatgaaca gaaatccccc ttacacggag gcatcagtga ccaaacagga aaaaaccgcc
     5761 cttaacatgg cccgctttat cagaagccag acattaacgc ttctggagaa actcaacgag
     5821 ctggacgcgg atgaacaggc agacatctgt gaatcgcttc acgaccacgc tgatgagctt
     5881 taccgcagct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     5941 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     6001 gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc
     6061 ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccatt
     6121 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc
     6181 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     6241 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     6301 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     6361 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     6421 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     6481 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     6541 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     6601 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     6661 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     6721 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     6781 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     6841 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     6901 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     6961 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgaa
     7021 caataaaact gtctgcttac a
//
