LOCUS       sequence-392768-        5989 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET28 driving expression of GFP, biobrick part Bba_E0040.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5989)
  AUTHORS   Shis DL, Bennett MR
  TITLE     Library of synthetic transcriptional AND gates built with split T7 
            RNA polymerase mutants.
  JOURNAL   Proc Natl Acad Sci U S A. 2013 Mar 26;110(13):5028-33. doi: 
            10.1073/pnas.1220157110. Epub 2013 Mar 11.
  PUBMED    23479654
REFERENCE   2  (bases 1 to 5989)
  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"; doi: 
            "10.1073/pnas.1220157110"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2013-03-26- 26"; volume: "110"; issue: "13"; pages: "5028-33"
COMMENT     Sequence Label: pET28:GFP
FEATURES             Location/Qualifiers
     source          1..5989
                     /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(204..917,918..920,921..923))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(207..917,918..920,921..923))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(249..270)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     598..617
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     857..878
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     RBS             930..952
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    967..991
                     /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(991..1010)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(992..1010)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1094..1113)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1319..1396
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1397..2479
                     /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(1416..1435)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3288..3479
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3473..3495)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3581..3720
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3636..3655
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3735..3752)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3906..4494)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3986..4005)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4605..4624)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4616..5431
                     /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             4616..5431
                     /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     complement(4689..4708)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5523..5978)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5660..5681)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5872..5891
                     /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 gagtgcggcc gcaagcttgt
      181 cgacggagct cgaattcgga tccttacttg tacagctcgt ccatgccgag agtgatcccg
      241 gcggcggtca cgaactccag caggaccatg tgatcgcgct tctcgttggg gtctttgctc
      301 agggcggact gggtgctcag gtagtggttg tcgggcagca gcacggggcc gtcgccgatg
      361 ggggtgttct gctggtagtg gtcggcgagc tgcacgctgc cgtcctcgat gttgtggcgg
      421 atcttgaagt tcaccttgat gccgttcttc tgcttgtcgg ccatgatata gacgttgtgg
      481 ctgttgtagt tgtactccag cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg
      541 cccttcagct cgatgcggtt caccagggtg tcgccctcga acttcacctc ggcgcgggtc
      601 ttgtagttgc cgtcgtcctt gaagaagatg gtgcgctcct ggacgtagcc ttcgggcatg
      661 gcggacttga agaagtcgtg ctgcttcatg tggtcggggt agcggctgaa gcactgcacg
      721 ccgtaggtca gggtggtcac gagggtgggc cagggcacgg gcagcttgcc ggtggtgcag
      781 atgaacttca gggtcagctt gccgtaggtg gcatcgccct cgccctcgcc ggacacgctg
      841 aacttgtggc cgtttacgtc gccgtccagc tcgaccagga tgggcaccac cccggtgaac
      901 agctcctcgc ccttgctcac catggtatat ctccttctta aagttaaaca aaattatttc
      961 tagaggggaa ttgttatccg ctcacaattc ccctatagtg agtcgtatta atttcgcggg
     1021 atcgagatct cgatcctcta cgccggacgc atcgtggccg gcatcaccgg cgccacaggt
     1081 gcggttgctg gcgcctatat cgccgacatc accgatgggg aagatcgggc tcgccacttc
     1141 gggctcatga gcgcttgttt cggcgtgggt atggtggcag gccccgtggc cgggggactg
     1201 ttgggcgcca tctccttgca tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac
     1261 ctactactgg gctgcttcct aatgcaggag tcgcataagg gagagcgtcg agatcccgga
     1321 caccatcgaa tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca
     1381 attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt
     1441 ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg
     1501 ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca
     1561 actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc
     1621 gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt
     1681 ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct
     1741 cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc
     1801 tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc
     1861 catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt
     1921 cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg
     1981 tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga
     2041 acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga
     2101 gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg
     2161 cgccattacc gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga
     2221 taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct
     2281 gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg
     2341 caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accctggcgc ccaatacgca
     2401 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     2461 actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact cattaggcac
     2521 cgggatctcg accgatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc
     2581 ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac
     2641 aggtgccggc agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga
     2701 tgatcggcct gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca
     2761 ctggtcccgc caccaaacgt ttcggcgaga agcaggccat tatcgccggc atggcggccc
     2821 cacgggtgcg catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc
     2881 ttactggtta gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca
     2941 aaacgtctgc gacctgagca acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc
     3001 tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg gatctgcatc gcaggatgct
     3061 gctggctacc ctgtggaaca cctacatctg tattaacgaa gcgctggcat tgaccctgag
     3121 tgatttttct ctggtcccgc cgcatccata ccgccagttg tttaccctca caacgttcca
     3181 gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag catcctctct cgtttcatcg
     3241 gtatcattac ccccatgaac agaaatcccc cttacacgga ggcatcagtg accaaacagg
     3301 aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca gacattaacg cttctggaga
     3361 aactcaacga gctggacgcg gatgaacagg cagacatctg tgaatcgctt cacgaccacg
     3421 ctgatgagct ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac
     3481 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag
     3541 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac
     3601 gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag
     3661 agtgcaccat tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
     3721 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     3781 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     3841 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     3901 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     3961 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     4021 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     4081 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     4141 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     4201 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     4261 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     4321 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca
     4381 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     4441 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     4501 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     4561 ttggtcatga acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag
     4621 ccatattcaa cgggaaacgt cttgctctag gccgcgatta aattccaaca tggatgctga
     4681 tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg
     4741 attgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc
     4801 caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc
     4861 gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc
     4921 cgggaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga
     4981 tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa
     5041 cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga
     5101 tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat
     5161 gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga
     5221 taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat
     5281 cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc
     5341 attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca
     5401 gtttcatttg atgctcgatg agtttttcta agaattaatt catgagcgga tacatatttg
     5461 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     5521 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     5581 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     5641 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     5701 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     5761 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     5821 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     5881 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     5941 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgcca
//
