LOCUS       sequence-59493-e        6669 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 6669)
  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 6669)
  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: pCoofy17
FEATURES             Location/Qualifiers
     source          1..6669
                     /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..150
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             160..177
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(424..426,427..1140)
                     /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(424..426,427..1137)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(466..487)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(727..746)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1074..1095
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     RBS             1213..1221
                     /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             1223..1531
                     /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     1410..1429
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1551..1568
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      1635..1682
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1719..2174
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1806..1825)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2016..2037
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2267..3082)
                     /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(2267..3082)
                     /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     2990..3009
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3074..3093
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3204..3792
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3693..3712
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3946..3963
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3978..4120
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4046..4065)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4206..4228
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4222..4413)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5222..6304)
                     /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     6266..6285
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6305..6382)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6588..6607
                     /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 atcatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat
      181 atgaatgacc acatcaacct gaaggtggcc gggcaggacg gctccgtggt gcagttcaag
      241 atcaagaggc acacgccgct gagcaagctg atgaaggcct actgcgagag gcagggcttg
      301 tcaatgaggc agatcagatt caggttcgac gggcagccaa tcaatgaaac tgacactcca
      361 gcacagctgg agatggagga cgaggacacc atcgacgtgt tccagcagca gaccggtgga
      421 tccgtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
      481 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
      541 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
      601 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
      661 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
      721 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
      781 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
      841 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
      901 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
      961 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     1021 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     1081 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct ctacaagtga
     1141 aagcttgata tcgaattcct gcagcccaaa ctgcagttga caacataaaa actttgtgtt
     1201 atacttgtaa cgtaaggagg taatgattca gtttaaggtt tacacctata aaagagagag
     1261 ccgttatcgt ctgtttgtgg atgtacagag tgatattatt gacacgcccg ggcgacggat
     1321 ggtgatcccc ctggccagtg cacgtctgct gtcagataaa gtctcccgtg aactttaccc
     1381 ggtggtgcat atcggggatg aaagctggcg catgatgacc accgatatgg ccagtgtgcc
     1441 ggtctccgtt atcggggaag aagtggctga tctcagccac cgcgaaaatg acatcaaaaa
     1501 cgccattaac ctgatgttct ggggaatata agcttgcggc cgcactcgag caccaccacc
     1561 accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca
     1621 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
     1681 tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt
     1741 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     1801 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     1861 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     1921 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     1981 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     2041 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     2101 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     2161 aacgtttaca atttcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     2221 atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat
     2281 cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa
     2341 aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat
     2401 cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct
     2461 cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga
     2521 atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt
     2581 catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac
     2641 gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca
     2701 ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct
     2761 ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga
     2821 taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct
     2881 catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat
     2941 cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc
     3001 atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg
     3061 tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt
     3121 ttattgttca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     3181 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     3241 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     3301 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     3361 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     3421 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     3481 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     3541 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     3601 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     3661 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     3721 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     3781 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     3841 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     3901 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     3961 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     4021 caccgcatat atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     4081 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     4141 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     4201 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     4261 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     4321 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     4381 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     4441 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     4501 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     4561 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     4621 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     4681 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     4741 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     4801 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     4861 cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa
     4921 acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac
     4981 cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac
     5041 ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc
     5101 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     5161 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     5221 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     5281 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     5341 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     5401 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     5461 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca
     5521 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     5581 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     5641 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     5701 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     5761 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     5821 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     5881 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     5941 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     6001 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     6061 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     6121 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     6181 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     6241 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     6301 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     6361 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt
     6421 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca
     6481 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg
     6541 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg
     6601 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
     6661 tagaggatc
//
