LOCUS       sequence-59507-e        5683 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 5683)
  AUTHORS   Scholz J, Besir H, Strasser C, Suppmann S
  TITLE     A new method to customize protein expression vectors for fast, 
            efficient and background free parallel cloning.
  JOURNAL   BMC Biotechnol. 2013 Feb 14;13(1):12.
  PUBMED    23410102
REFERENCE   2  (bases 1 to 5683)
  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: "BMC 
            Biotechnol."; date: "2013-02-14"; volume: "13(1)"; pages: "12"
COMMENT     Sequence Label: pCoofy40
FEATURES             Location/Qualifiers
     source          1..5683
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        42..421
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        422..625
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     576..596
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     622..646
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     1127..1146
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             1190..1213
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     RBS             1250..1258
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             1260..1568
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MIQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSD
                     KVSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1447..1466
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1572..1601
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             join(1605..1610,1611..1634,1635..1670,1671..1694)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             1698..1742
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             1749..1826
                     /codon_start=1
                     /product="calmodulin-binding peptide"
                     /label=CBP
                     /note="derived from skeletal muscle myosin light chain 
                     kinase; binds calmodulin with nanomolar affinity in the 
                     presence of calcium"
                     /translation="KRRWKKNFIAVSAANRFKKISSSGAL"
     CDS             1833..1868
                     /codon_start=1
                     /product="epitope tag derived from human Protein C (Stearns
                     et al., 1988)"
                     /label=Protein C tag
                     /translation="EDQVDPRLIDGK"
     polyA_signal    2559..2614
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2613..2632)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3697..3715)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3783..3887
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3888..3956,3957..4748)
                     /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(4106..4125)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4919..5507
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5408..5427
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5661..5678
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gtacatttat attggctcat gtccaatatg accgccatgt tgacattgat tattgactag
       61 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
      121 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
      181 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
      241 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
      301 tccgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
      361 gaccttacgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
      421 ggtgatgcgg ttttggcagt acaccaatgg gcgtggatag cggtttgact cacggggatt
      481 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
      541 ctttccaaaa tgtcgtaata accccgcccc gttgacgcaa atgggcggta ggcgtgtacg
      601 gtgggaggtc tatataagca gagctcgttt agtgaaccgt cagatcctca ctctcttccg
      661 catcgctgtc tgcgagggcc agctgttggg ctcgcggttg aggacaaact cttcgcggtc
      721 tttccagtac tcttggatcg gaaacccgtc ggcctccgaa cggtactccg ccaccgaggg
      781 acctgagcga gtccgcatcg accggatcgg aaaacctctc gagaaaggcg tctaaccagt
      841 cacagtcgca aggtaggctg agcaccgtgg cgggcggcag cgggtggcgg tcggggttgt
      901 ttctggcgga ggtgctgctg atgatgtaat taaagtaggc ggtcttgaga cggcggatgg
      961 tcgaggtgag gtgtggcagg cttgagatcc agctgttggg gtgagtactc cctctcaaaa
     1021 gcgggcatta cttctgcgct aagattgtca gtttccaaaa acgaggagga tttgatattc
     1081 acctggcccg atctggccat acacttgagt gacaatgaca tccactttgc ctttctctcc
     1141 acaggtgtcc actcccaggt ccaagtttaa acggatctct agcgaattcc tggaagttct
     1201 gttccagggg cccttgacaa cataaaaact ttgtgttata cttgtaacgt aaggaggtaa
     1261 tgattcagtt taaggtttac acctataaaa gagagagccg ttatcgtctg tttgtggatg
     1321 tacagagtga tattattgac acgcccgggc gacggatggt gatccccctg gccagtgcac
     1381 gtctgctgtc agataaagtc tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa
     1441 gctggcgcat gatgaccacc gatatggcca gtgtgccggt ctccgttatc ggggaagaag
     1501 tggctgatct cagccaccgc gaaaatgaca tcaaaaacgc cattaacctg atgttctggg
     1561 gaatataaga gcatcatcat catcaccacc accaccacca ctgaagcgct tggagccacc
     1621 cgcagttcga aaaaggtgga ggttctggcg gtggatcggg aggttcagcg tggagccacc
     1681 cgcagttcga gaaatagaaa gaaaccgctg ctgctaaatt cgaacgccag cacatggaca
     1741 gctagatgaa gcggcggtgg aagaaaaact ttatcgccgt gtccgccgcc aaccggttca
     1801 agaagatcag cagctctggc gccctgctgt gagaggacca ggtggacccc cggctgatcg
     1861 atggcaaatg aggcagcggc aagatcctgc acaaccagaa cgtgaacagc tggggcccca
     1921 tcaccgtgac ccctaccacc gacggcggcg agacaagatt cgacggccag atcatcgtgc
     1981 agatggaaaa cgaccccgtg gtggccaagg ccgctgccaa tctggccggc aagcacgccg
     2041 agagcagcgt ggtggtgcag ctggacagcg acggcaacta ccgggtggtg tacggcgacc
     2101 ccagcaagct ggacggcaag ctgcggtggc agctggtggg acacggcagg gaccacagcg
     2161 agacaaacaa cacccggctg agcggctaca gcgccgacga gctggccgtg aagctggcca
     2221 agttccagca gagcttcaac caggccgaga acatcaacaa caagcccgac cacatcagca
     2281 tcgtgggctg cagcctggtg tccgacgaca agcagaaggg cttcggccac cagttcatca
     2341 acgctatgga cgccaacggc ctgcgggtgg acgtgtccgt gcggtctagc gaactggccg
     2401 tggacgaggc cggcagaaag cacaccaagg acgccaatgg cgactgggtg cagaaggccg
     2461 aaaacaacaa ggtgtccctg agctgggacg cccagggcct ggaatgagcg gccgctcgag
     2521 gccggcaagg ccggatcccc cgacctcgac ctctggctaa taaaggaaat ttattttcat
     2581 tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga aggacatatg ggagggcaaa
     2641 tcatttggtc gagatccctc ggagatctct agctagagga tcgatccccg ccccggacga
     2701 actaaacctg actacgacat ctctgcccct tcttcgcggg gcagtgcatg taatcccttc
     2761 agttggttgg tacaacttgc caactgaacc ctaaacgggt agcatatgct tcccgggtag
     2821 tagtatatac tatccagact aaccctaatt caatagcata tgttacccaa cgggaagcat
     2881 atgctatcga attagggtta gtaaaagggt cctaaggaac agcgatgtag gtgggcgggc
     2941 caagataggg gcgcgattgc tgcgatctgg aggacaaatt acacacactt gcgcctgagc
     3001 gccaagcaca gggttgttgg tcctcatatt cacgaggtcg ctgagagcac ggtgggctaa
     3061 tgttgccatg ggtagcatat actacccaaa tatctggata gcatatgcta tcctaatcta
     3121 tatctgggta gcataggcta tcctaatcta tatctgggta gcatatgcta tcctaatcta
     3181 tatctgggta gtatatgcta tcctaattta tatctgggta gcataggcta tcctaatcta
     3241 tatctgggta gcatatgcta tcctaatcta tatctgggta gtatatgcta tcctaatctg
     3301 tatccgggta gcatatgcta tcctaataga gattagggta gtatatgcta tcctaattta
     3361 tatctgggta gcatatacta cccaaatatc tggatagcat atgctatcct aatctatatc
     3421 tgggtagcat atgctatcct aatctatatc tgggtagcat aggctatcct aatctatatc
     3481 tgggtagcat atgctatcct aatctatatc tgggtagtat atgctatcct aatttatatc
     3541 tgggtagcat aggctatcct aatctatatc tgggtagcat atgctatcct aatctatatc
     3601 tgggtagtat atgctatcct aatctgtatc cgggtagcat atgctatcct catgataagc
     3661 tgtcaaacat gagaattaat tcttgaagac gaaagggcct cgtgatacgc ctatttttat
     3721 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     3781 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     3841 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     3901 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     3961 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     4021 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     4081 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg
     4141 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     4201 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     4261 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     4321 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     4381 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg
     4441 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     4501 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     4561 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     4621 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     4681 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     4741 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     4801 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     4861 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     4921 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     4981 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     5041 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     5101 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     5161 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     5221 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     5281 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     5341 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     5401 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     5461 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     5521 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     5581 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     5641 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agc
//
