LOCUS       sequence-325035-        6116 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Tat(48-57) conjugated EGFP for protein purification 
            (6XHis tag).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6116)
  AUTHORS   Lee HM, Ren J, Tran KM, Jeon BM, Park WU, Kim H, Lee KE, Oh Y, Choi 
            M, Kim DS, Na D
  TITLE     Identification of efficient prokaryotic cell-penetrating peptides 
            with applications in bacterial biotechnology.
  JOURNAL   Commun Biol. 2021 Feb 15;4(1):205. doi: 10.1038/s42003-021-01726-w.
  PUBMED    33589718
REFERENCE   2  (bases 1 to 6116)
  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: "Commun 
            Biol."; date: "2021-02-15"; volume: "4(1):205. doi"; pages: " 
            10.1038/s42003-021-01726-w"
COMMENT     Sequence Label: pET21a.Tat(48-57)-EGFP
FEATURES             Location/Qualifiers
     source          1..6116
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(join(164..874,875..877))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             complement(join(164..874,875..877))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(206..227)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     555..574
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     814..835
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     RBS             924..946
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    961..985
                     /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)."
     primer_bind     complement(985..1004)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(986..1004)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1088..1107)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1313..1390
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1391..2473
                     /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     complement(1410..1429)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3282..3473
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3467..3489)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3575..3714
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3630..3649
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3729..3746)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3900..4488)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3980..3999)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4659..5450,5451..5519))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5282..5301
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5520..5624)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5650..6105)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5787..5808)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5999..6018
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagcttgtac agctcgtcca
      181 tgccgagagt gatcccggcg gcggtcacga actccagcag gaccatgtga tcgcgcttct
      241 cgttggggtc tttgctcagg gcggactggg tgctcaggta gtggttgtcg ggcagcagca
      301 cggggccgtc gccgatgggg gtgttctgct ggtagtggtc ggcgagctgc acgctgccgt
      361 cctcgatgtt gtggcggatc ttgaagttca ccttgatgcc gttcttctgc ttgtcggcca
      421 tgatatagac gttgtggctg ttgtagttgt actccagctt gtgccccagg atgttgccgt
      481 cctccttgaa gtcgatgccc ttcagctcga tgcggttcac cagggtgtcg ccctcgaact
      541 tcacctcggc gcgggtcttg tagttgccgt cgtccttgaa gaagatggtg cgctcctgga
      601 cgtagccttc gggcatggcg gacttgaaga agtcgtgctg cttcatgtgg tcggggtagc
      661 ggctgaagca ctgcacgccg taggtcaggg tggtcacgag ggtgggccag ggcacgggca
      721 gcttgccggt ggtgcagatg aacttcaggg tcagcttgcc gtaggtggca tcgccctcgc
      781 cctcgccgga cacgctgaac ttgtggccgt ttacgtcgcc gtccagctcg accaggatgg
      841 gcaccacccc ggtgaacagc tcctcgccct tgctcacgga tccacggcgc cgctgacggc
      901 gtttttttcg gcccatatgt atatctcctt cttaaagtta aacaaaatta tttctagagg
      961 ggaattgtta tccgctcaca attcccctat agtgagtcgt attaatttcg cgggatcgag
     1021 atctcgatcc tctacgccgg acgcatcgtg gccggcatca ccggcgccac aggtgcggtt
     1081 gctggcgcct atatcgccga catcaccgat ggggaagatc gggctcgcca cttcgggctc
     1141 atgagcgctt gtttcggcgt gggtatggtg gcaggccccg tggccggggg actgttgggc
     1201 gccatctcct tgcatgcacc attccttgcg gcggcggtgc tcaacggcct caacctacta
     1261 ctgggctgct tcctaatgca ggagtcgcat aagggagagc gtcgagatcc cggacaccat
     1321 cgaatggcgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag
     1381 ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta
     1441 tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa
     1501 agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc
     1561 gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc
     1621 gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc
     1681 gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca
     1741 acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga
     1801 agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa
     1861 cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt
     1921 gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg
     1981 tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga
     2041 aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat
     2101 cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat
     2161 taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga
     2221 agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg
     2281 gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca
     2341 gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc
     2401 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     2461 aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag gcaccgggat
     2521 ctcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca
     2581 tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc
     2641 cggcagcgct ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg
     2701 gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc
     2761 ccgccaccaa acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccccacggg
     2821 tgcgcatgat cgtgctcctg tcgttgagga cccggctagg ctggcggggt tgccttactg
     2881 gttagcagaa tgaatcaccg atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt
     2941 ctgcgacctg agcaacaaca tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa
     3001 cgcggaagtc agcgccctgc accattatgt tccggatctg catcgcagga tgctgctggc
     3061 taccctgtgg aacacctaca tctgtattaa cgaagcgctg gcattgaccc tgagtgattt
     3121 ttctctggtc ccgccgcatc cataccgcca gttgtttacc ctcacaacgt tccagtaacc
     3181 gggcatgttc atcatcagta acccgtatcg tgagcatcct ctctcgtttc atcggtatca
     3241 ttacccccat gaacagaaat cccccttaca cggaggcatc agtgaccaaa caggaaaaaa
     3301 ccgcccttaa catggcccgc tttatcagaa gccagacatt aacgcttctg gagaaactca
     3361 acgagctgga cgcggatgaa caggcagaca tctgtgaatc gcttcacgac cacgctgatg
     3421 agctttaccg cagctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc
     3481 agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc
     3541 agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg
     3601 atagcggagt gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca
     3661 ccattgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct
     3721 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     3781 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     3841 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     3901 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     3961 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     4021 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     4081 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     4141 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     4201 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     4261 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     4321 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
     4381 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     4441 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     4501 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     4561 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     4621 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     4681 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     4741 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     4801 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     4861 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     4921 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctgcaggc
     4981 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     5041 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     5101 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     5161 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     5221 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     5281 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     5341 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     5401 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     5461 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     5521 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     5581 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     5641 gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     5701 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     5761 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     5821 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     5881 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     5941 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     6001 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     6061 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgcca
//
