LOCUS       sequence-217199-        6486 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express 2(KW)2-eGFP in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6486)
  AUTHORS   Lee S, Oh Y, Lee J, Choe S, Lim S, Lee HS, Jo K, Schwartz DC
  TITLE     DNA binding fluorescent proteins for the direct visualization of 
            large DNA molecules.
  JOURNAL   Nucleic Acids Res. 2016 Jan 8;44(1):e6. doi: 10.1093/nar/gkv834. 
            Epub 2015 Aug 11.
  PUBMED    26264666
REFERENCE   2  (bases 1 to 6486)
  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: "Nucleic 
            Acids Res."; date: "2016-01-8"; volume: "44(1):e6. doi"; pages: " 
            10.1093/nar/gkv834. Epub 2015 Aug 11"
COMMENT     Sequence Label: pET-2(KW)2-eGFP
FEATURES             Location/Qualifiers
     source          1..6486
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(7..25)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        93..197
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(198..266,267..1058)
                     /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(416..435)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1229..1817
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1718..1737
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1971..1988
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2003..2142
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2068..2087)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2228..2250
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2244..2435)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3747..4829)
                     /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     4791..4810
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4830..4907)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5113..5132
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5216..5235
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5216..5234
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5235..5259
                     /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             5274..5296
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5315..5332
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5342..5359
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     regulatory      5405..5414
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(5411..5413,5414..5416,5417..6127)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFAYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5456..5477)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5717..5736)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6064..6085
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(6202..6220)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6216..6263
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(6438..6443,6444..6460,6461..6466))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
ORIGIN
        1 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
       61 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
      121 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
      181 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
      241 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
      301 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
      361 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
      421 gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact
      481 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
      541 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
      601 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
      661 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
      721 agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg
      781 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
      841 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
      901 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
      961 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     1021 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     1081 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     1141 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     1201 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     1261 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     1321 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     1381 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     1441 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     1501 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     1561 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     1621 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     1681 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     1741 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     1801 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     1861 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     1921 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     1981 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     2041 tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     2101 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
     2161 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     2221 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
     2281 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca
     2341 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg
     2401 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt
     2461 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact
     2521 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg
     2581 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta
     2641 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag
     2701 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt
     2761 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt
     2821 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg
     2881 agcacgatca tgcgcacccg tggccaggac ccaacgctgc ccgagatgcg ccgcgtgcgg
     2941 ctgctggaga tggcggacgc gatggatatg ttctgccaag ggttggtttg cgcattcaca
     3001 gttctccgca agaattgatt ggctccaatt cttggagtgg tgaatccgtt agcgaggtgc
     3061 cgccggcttc cattcaggtc gaggtggccc ggctccatgc accgcgacgc aacgcgggga
     3121 ggcagacaag gtatagggcg gcgcctacaa tccatgccaa cccgttccat gtgctcgccg
     3181 aggcggcata aatcgccgtg acgatcagcg gtccaatgat cgaagttagg ctggtaagag
     3241 ccgcgagcga tccttgaagc tgtccctgat ggtcgtcatc tacctgcctg gacagcatgg
     3301 cctgcaacgc gggcatcccg atgccgccgg aagcgagaag aatcataatg gggaaggcca
     3361 tccagcctcg cgtcgcgaac gccagcaaga cgtagcccag cgcgtcggcc gccatgccgg
     3421 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
     3481 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
     3541 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
     3601 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
     3661 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     3721 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     3781 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     3841 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     3901 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     3961 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     4021 accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     4081 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     4141 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     4201 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
     4261 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
     4321 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
     4381 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
     4441 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
     4501 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
     4561 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
     4621 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
     4681 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
     4741 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
     4801 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     4861 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
     4921 ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag
     4981 caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac
     5041 ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg
     5101 atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt
     5161 gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata
     5221 cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt
     5281 aactttaaga aggagatata ccatgggcag cagccatcat catcatcatc acagcagcgg
     5341 cctggtgccg cgcggcagcc atatgaaatg gaaatggaaa aaagcgatgc gtatcgattc
     5401 agccgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     5461 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     5521 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     5581 ctggcccacc ctcgtgacca ccttcgccta cggcctgcag tgcttcgccc gctaccccga
     5641 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     5701 caccatctcc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     5761 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     5821 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca ccgccgacaa
     5881 gcagaagaac ggcatcaagg ccaacttcaa gatccgccac aacatcgagg acggcagcgt
     5941 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     6001 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
     6061 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     6121 gtacaaggcg aaaaaatgga aatggaaata aggatccggc tgctaacaaa gcccgaaagg
     6181 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     6241 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggatat cccgcaagag
     6301 gcccggcagt accggcataa ccaagcctat gcctacagca tccagggtga cggtgccgag
     6361 gatgacgatg agcgcattgt tagatttcat acacggtgcc tgactgcgtt agcaatttaa
     6421 ctgtgataaa ctaccgcatt aaagcttatc gatgataagc tgtcaaacat gagaattctt
     6481 gaagac
//
