LOCUS       sequence-217201-        6514 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express 2HMG-eGFP in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6514)
  AUTHORS   Lee Seonghyun, Cong Wang, Junghyun Song, Do-geun Kim, Yeeun Oh, 
            Wooseok Ko, Jinyong Lee, Jungyul Park, Hyun Soo Lee, Kyubong Jo
  TITLE     Investigation of various fluorescent protein–DNA binding peptides 
            for effectively visualizing large DNA molecules
  JOURNAL   RSC Adv, 2016, 6, 46291-46298
REFERENCE   2  (bases 1 to 6514)
  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: "RSC Adv, 
            2016, 6, 46291-46298"
COMMENT     Sequence Label: pET-2HMG-eGFP
FEATURES             Location/Qualifiers
     source          1..6514
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        80..184
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(185..253,254..1045)
                     /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(403..422)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1216..1804
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1705..1724
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1958..1975
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1990..2129
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2055..2074)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2215..2237
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2231..2422)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3734..4816)
                     /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     4778..4797
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4817..4894)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5100..5119
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5203..5222
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5203..5221
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5222..5246
                     /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             5261..5283
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5302..5319
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5329..5346
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(5401..5403,5404..5406,5407..6117)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFAYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5446..5467)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5707..5726)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6054..6075
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(6217..6235)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6231..6278
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(6453..6458,6459..6475,6476..6481))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(join(6508..6514,1..12))
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
       61 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
      121 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
      181 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
      241 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
      301 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
      361 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
      421 atcccgtgtt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
      481 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
      541 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
      601 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
      661 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
      721 gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
      781 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
      841 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
      901 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
      961 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     1021 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     1081 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     1141 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     1201 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     1261 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     1321 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     1381 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     1441 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     1501 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     1561 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     1621 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     1681 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     1741 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     1801 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     1861 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     1921 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     1981 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa
     2041 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     2101 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     2161 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     2221 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa
     2281 gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct
     2341 cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg
     2401 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg
     2461 taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg
     2521 cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga
     2581 gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg
     2641 gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc
     2701 gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg
     2761 cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct
     2821 aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc
     2881 gcacccgtgg ccaggaccca acgctgcccg agatgcgccg cgtgcggctg ctggagatgg
     2941 cggacgcgat ggatatgttc tgccaagggt tggtttgcgc attcacagtt ctccgcaaga
     3001 attgattggc tccaattctt ggagtggtga atccgttagc gaggtgccgc cggcttccat
     3061 tcaggtcgag gtggcccggc tccatgcacc gcgacgcaac gcggggaggc agacaaggta
     3121 tagggcggcg cctacaatcc atgccaaccc gttccatgtg ctcgccgagg cggcataaat
     3181 cgccgtgacg atcagcggtc caatgatcga agttaggctg gtaagagccg cgagcgatcc
     3241 ttgaagctgt ccctgatggt cgtcatctac ctgcctggac agcatggcct gcaacgcggg
     3301 catcccgatg ccgccggaag cgagaagaat cataatgggg aaggccatcc agcctcgcgt
     3361 cgcgaacgcc agcaagacgt agcccagcgc gtcggccgcc atgccggcga taatggcctg
     3421 cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa
     3481 gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc
     3541 gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac
     3601 agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt
     3661 gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt
     3721 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     3781 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc
     3841 ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt
     3901 gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta
     3961 acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg
     4021 caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt
     4081 tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa
     4141 aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag
     4201 tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg
     4261 ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac
     4321 cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata
     4381 acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat
     4441 agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg
     4501 cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc
     4561 gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa
     4621 cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat
     4681 tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct
     4741 ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata
     4801 acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca
     4861 taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc
     4921 gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca
     4981 aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc
     5041 atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg
     5101 gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg
     5161 atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag
     5221 gggaattgtg agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg
     5281 agatatacca tgggcagcag ccatcatcat catcatcaca gcagcggcct ggtgccgcgc
     5341 ggcagccata tgaccccgaa acgcccgcgc ggccgcccga aaaaaggcgg cagcggcggc
     5401 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     5461 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     5521 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     5581 ctcgtgacca ccttcgccta cggcctgcag tgcttcgccc gctaccccga ccacatgaag
     5641 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatctcc
     5701 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     5761 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     5821 aagctggagt acaactacaa cagccacaac gtctatatca ccgccgacaa gcagaagaac
     5881 ggcatcaagg ccaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     5941 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     6001 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     6061 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagggc
     6121 ggcagcggcg gcaaaaaacc gcgcggccgc ccgcgcaaac cgacctagga tccgctgcta
     6181 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
     6241 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     6301 gatatcccgc aagaggcccg gcagtaccgg cataaccaag cctatgccta cagcatccag
     6361 ggtgacggtg ccgaggatga cgatgagcgc attgttagat ttcatacacg gtgcctgact
     6421 gcgttagcaa tttaactgtg ataaactacc gcattaaagc ttatcgatga taagctgtca
     6481 aacatgagaa ttcttgaaga cgaaagggcc tcgt
//
