LOCUS       sequence-46246-e        5676 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 5676)
  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 5676)
  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: "Nat 
            Protoc."; date: "2010-08"; volume: "5(8)"; pages: "1447-59. Epub 
            2010 Jul 22"
COMMENT     Sequence Label: pEBB-BT
FEATURES             Location/Qualifiers
     source          1..5676
                     /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      1788..1797
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2049..2069
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(2810..2827)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2810..2826)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2819..2841)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3039..3494
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3126..3145)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3336..3357
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3511..3530)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3630..3652
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3690..3708)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3776..3880
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3881..3949,3950..4741)
                     /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(4099..4118)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4912..5500
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5401..5420
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5654..5671
                     /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 gaattctcta gagatccctc gacctcgaga tccattgtgc tggatctgcc accatggctg
     1801 gaaaggccgg tgaaggtgaa atccctgccc ctcttgctgg taccgtttct aagatactgg
     1861 taaaagaagg tgacactgtt aaagctggtc aaacagttct ggtgctggag gctatgaaaa
     1921 tggagacaga aattaacgct cctactgacg gaaaagttga aaaggtgtta gttaaggaaa
     1981 gagatgctgt tcaaggtggt caaggtctaa tcaagatcgg cgactacgat atacccacaa
     2041 ccgccagcga gaatctgtac tttcagagtt aaaaaccgcc gcactagctc aacatgacga
     2101 ggctggatcc actagtggta ccatcgatgc ggccgcgact ctagagtgag ggtccccacc
     2161 tgggaccctt gagagtatca ggtctcccac gtgggagaca agaaatccct gtttaatatt
     2221 taaacagcag tgttccccat ctgggtcctt gcacccctca ctctggcctc agccgactgc
     2281 acagcggccc ctgcatcccc ttggctgtga ggcccctgga caagcagagg tggccagagc
     2341 tgggaggcat ggccctgggg tcccacgaat ttgctgggga atctcgtttt tcttcttaag
     2401 acttttggga catggtttga ctcccgaaca tcaccgacgt gtctcctgtt tttctgggtg
     2461 gcctcgggac acctgccctg cccccacgag ggtcaggact gtgactcttt ttagggccag
     2521 gcaggtgcct ggacatttgc cttgctggac ggggactggg gatgtgggag ggagcagaca
     2581 ggaggaatca tgtcaggcct gtgtgtgaaa ggaagctcca ctgtcaccct ccacctcttc
     2641 accccccact caccagtgtc ccctccactg tcacattgta actgaacttc aggataataa
     2701 agtgtttgcc tccaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     2761 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaagaattca ctggccgtcg
     2821 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     2881 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     2941 agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt
     3001 gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt
     3061 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     3121 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     3181 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     3241 caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     3301 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     3361 aacactcaac cctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggc
     3421 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     3481 aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     3541 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     3601 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     3661 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     3721 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     3781 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     3841 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     3901 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     3961 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     4021 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     4081 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     4141 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     4201 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     4261 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     4321 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     4381 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     4441 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     4501 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     4561 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     4621 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     4681 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     4741 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     4801 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     4861 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     4921 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     4981 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     5041 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     5101 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     5161 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     5221 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     5281 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     5341 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     5401 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     5461 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     5521 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     5581 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     5641 aacgaccgag cgcagcgagt cagtgagcga ggaagc
//
