LOCUS       sequence-44713-e        5892 bp    DNA     circular SYN 15-APR-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5892)
  AUTHORS   Maine GN, Li H, Zaidi IW, Basrur V, Elenitoba-Johnson KS, Burstein E
  TITLE     A bimolecular affinity purification method under denaturing 
            conditions for rapid isolation of a ubiquitinated protein for mass 
            spectrometry analysis.
  JOURNAL   Nat Protoc. 2010 Aug;5(8):1447-59. Epub 2010 Jul 22.
  PUBMED    20671728
REFERENCE   2  (bases 1 to 5892)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Protoc."; date: "2010-08"; volume: "5(8)"; pages: "1447-59. Epub 
            2010 Jul 22"
COMMENT     Sequence Label: pEBB-BT-Ubiquitin
FEATURES             Location/Qualifiers
     source          1..5892
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        252..462
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        274..470
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      321..456
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        557..1739
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          788..1730
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1687..1707
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1787..1796
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2048..2068
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2114..2341
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     primer_bind     complement(3026..3043)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3026..3042)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3035..3057)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3255..3710
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3342..3361)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3552..3573
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3727..3746)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3846..3868
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3906..3924)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3992..4096
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4097..4165,4166..4957)
                     /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(4315..4334)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5128..5716
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5617..5636
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5870..5887
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttc
      241 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
      301 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
      361 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc
      421 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctttgca
      481 aagatggata aagttttaaa cagagaggaa tctttgcagc taatggacct tctaggtctt
      541 gaaaggagtg ggaattggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt
      601 ccccgagaag ttggggggag gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg
      661 ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga
      721 accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag
      781 aacacaggta agtgccgtgt gtggttcccg cgggcctggc ctctttacgg gttatggccc
      841 ttgcgtgcct tgaattactt ccacgcccct ggctgcagta cgtgattctt gatcccgagc
      901 ttcgggttgg aagtgggtgg gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc
      961 gtgcttgagt tgaggcctgg cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc
     1021 ttcgcgcctg tctcgctgct ttcgataagt ctctagccat ttaaaatttt tgatgacctg
     1081 ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc gggccaagat ctgcacactg
     1141 gtatttcggt ttttggggcc gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt
     1201 cggcgaggcg gggcctgcga gcgcggccac cgagaatcgg acgggggtag tctcaagctg
     1261 gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa
     1321 ggctggcccg gtcggcacca gttgcgtgag cggaaagatg gccgcttccc ggccctgctg
     1381 cagggagctc aaaatggagg acgcggcgct cgggagagcg ggcgggtgag tcacccacac
     1441 aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg tgactccacg gagtaccggg
     1501 cgccgtccag gcacctcgat tagttctcga gcttttggag tacgtcgtct ttaggttggg
     1561 gggaggggtt ttatgcgatg gagtttcccc acactgagtg ggtggagact gaagttaggc
     1621 cagcttggca cttgatgtaa ttctccttgg aatttgccct ttttgagttt ggatcttggt
     1681 tcattctcaa gcctcagaca gtggttcaaa gtttttttct tccatttcag gtgtcgtgag
     1741 gattctctag agatccctcg acctcgagat ccattgtgct ggatctgcca ccatggctgg
     1801 aaaggccggt gaaggtgaaa tccctgcccc tcttgctggt accgtttcta agatactggt
     1861 aaaagaaggt gacactgtta aagctggtca aacagttctg gtgctggagg ctatgaaaat
     1921 ggagacagaa attaacgctc ctactgacgg aaaagttgaa aaggtgttag ttaaggaaag
     1981 agatgctgtt caaggtggtc aaggtctaat caagatcggc gactacgata tacccacaac
     2041 cgccagcgag aatctgtact ttcaatcaga gttaaaaacc gccgcactag ctcaacatga
     2101 cgaggctgga tccatgcaga ttttcgtgaa aacccttacg gggaagacca tcaccctcga
     2161 ggttgaaccc tcggatacga tagaaaatgt aaaggccaag atccaggata aggaaggaat
     2221 tcctcctgat cagcagagac tgatctttgc tggcaagcag ctggaagatg gacgtacttt
     2281 gtctgactac aatattcaaa aggagtctac tcttcatctt gtgttgagac ttcgtggtgg
     2341 ttaagcggcc gcgactctag agtgagggtc cccacctggg acccttgaga gtatcaggtc
     2401 tcccacgtgg gagacaagaa atccctgttt aatatttaaa cagcagtgtt ccccatctgg
     2461 gtccttgcac ccctcactct ggcctcagcc gactgcacag cggcccctgc atccccttgg
     2521 ctgtgaggcc cctggacaag cagaggtggc cagagctggg aggcatggcc ctggggtccc
     2581 acgaatttgc tggggaatct cgtttttctt cttaagactt ttgggacatg gtttgactcc
     2641 cgaacatcac cgacgcgtct cctgtttttc tgggtggcct cgggacacct gccctgcccc
     2701 cacgagggtc aggactgtga ctctttttag ggccaggcag gtgcctggac atttgccttg
     2761 ctggacgggg actggggatg tgggagggag cagacaggag gaatcatgtc aggcctgtgt
     2821 gtgaaaggaa gctccactgt caccctccac ctcttcaccc cccactcacc agtgtcccct
     2881 ccactgtcac attgtaactg aacttcagga taataaagtg tttgcctcca aaaaaaaaaa
     2941 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     3001 aaaaaaaaaa aaaaaaaaag aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     3061 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     3121 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     3181 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc
     3241 aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     3301 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     3361 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     3421 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg
     3481 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     3541 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcgggcta
     3601 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     3661 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac
     3721 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     3781 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     3841 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
     3901 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     3961 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     4021 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     4081 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     4141 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     4201 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     4261 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     4321 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     4381 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     4441 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     4501 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     4561 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     4621 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     4681 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     4741 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     4801 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     4861 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     4921 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     4981 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     5041 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     5101 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     5161 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     5221 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     5281 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     5341 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     5401 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     5461 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     5521 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     5581 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     5641 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     5701 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     5761 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     5821 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     5881 gagcgaggaa gc
//
