LOCUS       sequence-246107-        5993 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5993)
  AUTHORS   Chen B, Mariano J, Tsai YC, Chan AH, Cohen M, Weissman AM
  TITLE     The activity of a human endoplasmic reticulum-associated degradation
            E3, gp78, requires its Cue domain, RING finger, and an E2-binding 
            site.
  JOURNAL   Proc Natl Acad Sci U S A. 2006 Jan 10. 103(2):341-6.
  PUBMED    16407162
REFERENCE   2  (bases 1 to 5993)
  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: "Proc Natl 
            Acad Sci U S A. 2006 Jan 10. 103(2):341-6."
COMMENT     Sequence Label: pGEX4T2 gp78 CD-m1,2 / JM25
FEATURES             Location/Qualifiers
     source          1..5993
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(85..90,91..106,107..113)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    121..137
                     /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)."
     CDS             160..813
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     771..793
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             820..837
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(1945..1967)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2112..2130)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2198..2302
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2303..2371,2372..3163)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2521..2540)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3334..3922
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3823..3842
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4076..4093
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4166..4243
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4244..5326
                     /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(4263..4282)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5375..5380,5381..5398,5399..5405)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5413..5429
                     /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     5437..5453
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5437..5453
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5449..5976
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5469..5486)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5469..5485)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5478..5500)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 gtcgctcaag gcgcactccc gttctggata atgttttttg cgccgacatc ataacggttc
       61 tggcaaatat tctgaaatga gctgttgaca attaatcatc ggctcgtata atgtgtggaa
      121 ttgtgagcgg ataacaattt cacacaggaa acagtattca tgtcccctat actaggttat
      181 tggaaaatta agggccttgt gcaacccact cgacttcttt tggaatatct tgaagaaaaa
      241 tatgaagagc atttgtatga gcgcgatgaa ggtgataaat ggcgaaacaa aaagtttgaa
      301 ttgggtttgg agtttcccaa tcttccttat tatattgatg gtgatgttaa attaacacag
      361 tctatggcca tcatacgtta tatagctgac aagcacaaca tgttgggtgg ttgtccaaaa
      421 gagcgtgcag agatttcaat gcttgaagga gcggttttgg atattagata cggtgtttcg
      481 agaattgcat atagtaaaga ctttgaaact ctcaaagttg attttcttag caagctacct
      541 gaaatgctga aaatgttcga agatcgttta tgtcataaaa catatttaaa tggtgatcat
      601 gtaacccatc ctgacttcat gttgtatgac gctcttgatg ttgttttata catggaccca
      661 atgtgcctgg atgcgttccc aaaattagtt tgttttaaaa aacgtattga agctatccca
      721 caaattgata agtacttgaa atccagcaag tatatagcat ggcctttgca gggctggcaa
      781 gccacgtttg gtggtggcga ccatcctcca aaatcggatc tggttccgcg tggatcccca
      841 ggaattcgtc ggcacaagaa ctatctacgt gtggttggaa acatggaggc caggtttgca
      901 gttgcaactc cagaggagct ggctgtcaac aatgacgact gtgtcatctg ttgggactcc
      961 atgcaggctg cgcggaaact gccctgtgga catcttttcc acaactcctg tcttcgttcc
     1021 tggctagaac aagacacctc ctgtccaaca tgcagaatgt ctcttaatat tgccgacaat
     1081 aatcgtgtca gggaagaaca tcaaggagag aacttggatg agaatttggt tcctgtagca
     1141 gcagccgaag ggagacctcg cttaaaccaa cacaatcact tcttccattt cgatgggtct
     1201 cggattgcga gctggctgcc gagtttttcg gttgaagtga tgcacaccac caacattctt
     1261 ggcattacgc aggccagcaa ctcccagctc aatgcaatgg ctcatcagat tcaagagggc
     1321 ggccgccagg ttccatacca tctgcgactg caggtcgacc agctgacacg ctcagttgaa
     1381 ataacaacag acaatatttt agaaggacgg attcaagtac cttttcctac acagcggtca
     1441 gatagcatca gacctgcatt gaacagtcct gtggaaaggc caagcagtga ccaggaagag
     1501 ggagaaactt ctgctcagac cgagcgtgtg ccactggacc tcagtcctcg cctggaggag
     1561 acgctggact tcggcgaggt ggaagtggag cccagtgagg tggaagactt cgaggctcgt
     1621 gggagccgct tctccaagtc tgctgatgag agacagcgca tgctggtgca gcgtaaggac
     1681 gaactcctcc agcaagctcg caaacgtttc ttgaacaaaa gttctgaaga tgatgcggcc
     1741 tcagagagct tcctcccctc ggaaggtgcg tcctctgacc ccgtgaccct gcgtcgaagg
     1801 atgctggctg ccgccgcgga acggaggctt cagaagcagc agacctccta gcgctccctt
     1861 gccttcctca gctggtcgac tcgagcggcc gcatcgtgac tgactgacga tctgcctcgc
     1921 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     1981 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     2041 cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt ataattcttg
     2101 aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     2161 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     2221 tttctaaata cattcaaata tgtatccgct catgagacaa caaccctgat aaatgcttca
     2281 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     2341 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     2401 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     2461 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     2521 gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat
     2581 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     2641 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     2701 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     2761 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     2821 cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac
     2881 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     2941 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     3001 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     3061 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     3121 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     3181 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     3241 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     3301 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     3361 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     3421 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     3481 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     3541 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     3601 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     3661 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     3721 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     3781 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     3841 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     3901 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     3961 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     4021 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     4081 gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg
     4141 cggtatttca caccgcataa attccgacac catcgaatgg cgcaaaacct ttcgcggtat
     4201 ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt
     4261 atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca
     4321 ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa
     4381 ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt
     4441 tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg
     4501 cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc
     4561 ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta
     4621 tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt
     4681 atttcttgat gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg
     4741 tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc
     4801 gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac
     4861 tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt
     4921 tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa
     4981 cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc
     5041 ggatatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt
     5101 aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca
     5161 actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag
     5221 aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     5281 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     5341 atgtaagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
     5401 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
     5461 acggattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     5521 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     5581 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgctt tgcctggttt
     5641 ccggcaccag aagcggtgcc ggaaagctgg ctggagtgcg atcttcctga ggccgatact
     5701 gtcgtcgtcc cctcaaactg gcagatgcac ggttacgatg cgcccatcta caccaacgtg
     5761 acctatccca ttacggtcaa tccgccgttt gttcccacgg agaatccgac gggttgttac
     5821 tcgctcacat ttaatgttga tgaaagctgg ctacaggaag gccagacgcg aattattttt
     5881 gatggcgttg gaattagctt atcgactgca cggtgcacca atgcttctgg cgtcaggcag
     5941 ccatcggaag ctgtggtatg gctgtgcagg tcgtaaatca ctgcataatt cgt
//
