LOCUS       sequence-59492-e        6657 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 6657)
  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 6657)
  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: pCoofy6
FEATURES             Location/Qualifiers
     source          1..6657
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     22..41
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        22..40
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    41..65
                     /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)."
     RBS             80..102
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             121..138
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             148..165
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(412..414,415..1128)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(412..414,415..1125)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(454..475)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(715..734)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1062..1083
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     RBS             1201..1209
                     /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             1211..1519
                     /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     1398..1417
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1539..1556
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      1623..1670
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1707..2162
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1794..1813)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2004..2025
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2255..3070)
                     /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             complement(2255..3070)
                     /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     2978..2997
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3062..3081
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3192..3780
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3681..3700
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3934..3951
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3966..4108
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4034..4053)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4194..4216
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4210..4401)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5210..6292)
                     /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     6254..6273
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6293..6370)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6576..6595
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gagatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca
       61 attcccctct agaaataatt ttgtttaact ttaagaagga gatataccat gggcagcagc
      121 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccatat gaatgaccac
      181 atcaacctga aggtggccgg gcaggacggc tccgtggtgc agttcaagat caagaggcac
      241 acgccgctga gcaagctgat gaaggcctac tgcgagaggc agggcttgtc aatgaggcag
      301 atcagattca ggttcgacgg gcagccaatc aatgaaactg acactccagc acagctggag
      361 atggaggacg aggacaccat cgacgtgttc cagcagcaga ccggtggatc cgtgagcaag
      421 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
      481 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
      541 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
      601 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
      661 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
      721 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
      781 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
      841 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
      901 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
      961 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     1021 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     1081 gtgaccgccg ccgggatcac tctcggcatg gacgagctct acaagtgaaa gcttgatatc
     1141 gaattcctgc agcccaaact gcagttgaca acataaaaac tttgtgttat acttgtaacg
     1201 taaggaggta atgattcagt ttaaggttta cacctataaa agagagagcc gttatcgtct
     1261 gtttgtggat gtacagagtg atattattga cacgcccggg cgacggatgg tgatccccct
     1321 ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa ctttacccgg tggtgcatat
     1381 cggggatgaa agctggcgca tgatgaccac cgatatggcc agtgtgccgg tctccgttat
     1441 cggggaagaa gtggctgatc tcagccaccg cgaaaatgac atcaaaaacg ccattaacct
     1501 gatgttctgg ggaatataag cttgcggccg cactcgagca ccaccaccac caccactgag
     1561 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
     1621 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
     1681 gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     1741 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     1801 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     1861 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     1921 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     1981 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     2041 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     2101 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
     2161 ttcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     2221 acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat
     2281 gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
     2341 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
     2401 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
     2461 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt
     2521 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
     2581 tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat
     2641 cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
     2701 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
     2761 tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
     2821 tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat
     2881 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
     2941 acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
     3001 ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa
     3061 tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg
     3121 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     3181 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     3241 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     3301 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     3361 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     3421 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     3481 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     3541 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     3601 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     3661 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     3721 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     3781 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     3841 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     3901 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     3961 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatat
     4021 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
     4081 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     4141 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     4201 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag
     4261 ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc
     4321 gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc
     4381 ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt
     4441 aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc
     4501 ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag
     4561 aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg
     4621 tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg
     4681 cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc
     4741 agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta
     4801 accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg
     4861 cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc
     4921 gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag
     4981 gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc
     5041 cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt
     5101 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
     5161 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     5221 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     5281 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     5341 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     5401 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     5461 cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg
     5521 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     5581 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     5641 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     5701 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     5761 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     5821 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     5881 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     5941 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     6001 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     6061 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     6121 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     6181 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     6241 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     6301 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
     6361 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc
     6421 agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg
     6481 gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc
     6541 atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca
     6601 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatc
//
