LOCUS       sequence-243826-        6468 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E.coli vector for parallel SLIC cloning containing N-terminal 
            His6-eGFP with PreScission Cleavage site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6468)
  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 6468)
  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: pCoofy61
FEATURES             Location/Qualifiers
     source          1..6468
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     26..45
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     129..148
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        129..147
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    148..172
                     /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             187..209
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             222..239
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(240..242,243..953)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLKE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(282..303)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(543..562)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             984..1007
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     RBS             1123..1131
                     /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             1133..1441
                     /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     1320..1339
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1461..1478
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1531..1549)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1545..1592
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1629..2084
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1716..1735)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1926..1947
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2176..2991)
                     /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(2176..2991)
                     /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     2899..2918
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2983..3002
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3113..3701
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3602..3621
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3855..3872
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3887..4026
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3952..3971)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4112..4134
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4128..4319)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5128..6210)
                     /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     6172..6191
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6211..6288)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
       61 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc
      121 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
      181 aataattttg tttaacttta agaaggagat ataccatgaa acatcaccat caccatcacg
      241 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
      301 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
      361 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
      421 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
      481 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
      541 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
      601 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
      661 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
      721 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
      781 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
      841 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctta
      901 aggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aaggggggtg
      961 gtggctccgg tggcggaggt accctggaag ttctgttcca ggggcccatg gactaatgag
     1021 gtaccggatc cgaattcgag ctccgtcgac aagcttgata tcgaattcct gcagcccaaa
     1081 ctgcagttga caacataaaa actttgtgtt atacttgtaa cgtaaggagg taatgattca
     1141 gtttaaggtt tacacctata aaagagagag ccgttatcgt ctgtttgtgg atgtacagag
     1201 tgatattatt gacacgcccg ggcgacggat ggtgatcccc ctggccagtg cacgtctgct
     1261 gtcagataaa gtctcccgtg aactttaccc ggtggtgcat atcggggatg aaagctggcg
     1321 catgatgacc accgatatgg ccagtgtgcc ggtctccgtt atcggggaag aagtggctga
     1381 tctcagccac cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct ggggaatata
     1441 agcttgcggc cgcactcgag caccaccacc accaccactg agatccggct gctaacaaag
     1501 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
     1561 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg
     1621 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     1681 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     1741 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     1801 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     1861 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     1921 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     1981 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     2041 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     2101 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     2161 ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat
     2221 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc
     2281 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc
     2341 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
     2401 accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac
     2461 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt
     2521 attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt
     2581 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc
     2641 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt
     2701 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa
     2761 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt
     2821 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc
     2881 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt
     2941 ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct
     3001 tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg
     3061 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     3121 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     3181 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     3241 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     3301 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     3361 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     3421 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     3481 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     3541 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     3601 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     3661 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     3721 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     3781 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     3841 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     3901 ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg
     3961 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     4021 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     4081 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     4141 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     4201 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     4261 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     4321 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
     4381 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
     4441 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
     4501 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
     4561 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
     4621 cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
     4681 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
     4741 cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg
     4801 gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga
     4861 gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag
     4921 cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc
     4981 atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag
     5041 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
     5101 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     5161 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     5221 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
     5281 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     5341 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
     5401 taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     5461 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     5521 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     5581 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     5641 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     5701 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
     5761 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     5821 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     5881 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     5941 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     6001 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     6061 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     6121 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     6181 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     6241 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac
     6301 gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga
     6361 gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca
     6421 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccga
//
