LOCUS       sequence-206010-        6121 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 6121)
  AUTHORS   Hsieh JM, Besserer GM, Madej MG, Bui HQ, Kwon S, Abramson J
  TITLE     Bridging the gap: a GFP-based strategy for overexpression and 
            purification of membrane proteins with intra and extracellular 
            C-termini.
  JOURNAL   Protein Sci. 2010 Apr;19(4):868-80.
  PUBMED    20196076
REFERENCE   2  (bases 1 to 6121)
  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: "Protein 
            Sci."; date: "2010-04"; volume: "19(4)"; pages: "868-80"
COMMENT     Sequence Label: pWarf(+)
FEATURES             Location/Qualifiers
     source          1..6121
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     921..940
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(960..1037)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1243..1262
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1346..1365
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1346..1364
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1365..1389
                     /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             1404..1426
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1461..1484
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1602..2312
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKAN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(1629..1657)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2252..2272
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             2325..2348
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(2401..2419)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2415..2462
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2499..2954
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2586..2605)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2796..2817
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3046..3861)
                     /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(3046..3861)
                     /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     3769..3788
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3853..3872
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3983..4571
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4472..4491
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4725..4742
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4757..4896
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4822..4841)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4982..5004
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4998..5189)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5998..6121,1..959))
                     /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"
ORIGIN
        1 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
       61 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
      121 gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg
      181 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
      241 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
      301 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
      361 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
      421 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
      481 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
      541 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
      601 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
      661 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
      721 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
      781 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
      841 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
      901 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
      961 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     1021 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     1081 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     1141 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     1201 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     1261 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     1321 gatcgagatc tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat
     1381 aacaattccc ctctagaaat aattttgttt aactttaaga aggagactcg agatgttaag
     1441 aattccgggt acctggatcc ttggaagtct tgtttcaggg accattaatt aacggtggtg
     1501 agattacatt aattattttt ggtgtgatgg ctggtgttat tggaacgatc ctcttaattt
     1561 cttacggtat tcgtcgtctg attaagggtt ctggtggttt cagcaaagga gaagaacttt
     1621 tcactggagt tgtcccaatt cttgttgaat tagatggtga tgttaatggg cacaaatttt
     1681 ctgtcagtgg agagggtgaa ggtgatgcta catacggaaa actcaccctt aaatttattt
     1741 gcactactgg aaaactacct gttccatggc caacacttgt cactactctg acctatggtg
     1801 ttcaatgctt ttcccgttat ccggatcaca tgaaacggca tgactttttc aagagtgcca
     1861 tgcccgaagg ttatgtacag gaacgcacta tatctttcaa agatgacggg aactacaaga
     1921 cgcgtgctga agtcaagttt gaaggtgata cccttgttaa tcgtatcgag ttaaagggta
     1981 ttgattttaa agaagatgga aacattctcg gacacaaact agagtacaac tataactcac
     2041 acaatgtata catcacggca gacaaacaaa agaatggaat caaagctaac ttcaaaattc
     2101 gccacaacat tgaagatggt tccgttcaac tagcagacca ttatcaacaa aatactccaa
     2161 ttggcgatgg ccctgtcctt ttaccagaca accattacct gtcgacacaa tctgcccttt
     2221 cgaaagatcc caacgaaaag cgtgaccaca tggtccttct tgagtttgta actgctgctg
     2281 ggattacaca tggcatggat gagctctaca aaaagcttgc ggcccatcat catcaccacc
     2341 accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca
     2401 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
     2461 tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt
     2521 aagcacggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     2581 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     2641 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     2701 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     2761 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     2821 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     2881 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     2941 aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     3001 tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc
     3061 gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa
     3121 aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc
     3181 ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc
     3241 gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa
     3301 tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc
     3361 atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg
     3421 aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag
     3481 gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg
     3541 gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat
     3601 aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc
     3661 atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc
     3721 gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca
     3781 tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt
     3841 ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag cagacagttt
     3901 tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     3961 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     4021 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     4081 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     4141 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     4201 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     4261 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     4321 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     4381 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     4441 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     4501 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     4561 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     4621 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     4681 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     4741 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     4801 accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     4861 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     4921 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     4981 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     5041 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
     5101 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     5161 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     5221 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     5281 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
     5341 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     5401 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     5461 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     5521 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     5581 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     5641 atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt
     5701 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca
     5761 agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag
     5821 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg
     5881 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
     5941 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     6001 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     6061 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     6121 g
//
