LOCUS       sequence-426370-        4377 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of I3-01.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4377)
  TITLE     Cheeseman lab plasmids Nov 2023
REFERENCE   2  (bases 1 to 4377)
  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"
COMMENT     Sequence Label: pGS140_6xHis-EGFP-I3-01
FEATURES             Location/Qualifiers
     source          1..4377
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     20..39
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        20..38
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             70..92
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             112..129
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             133..165
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(169..177,178..192)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(235..237,238..240,241..951)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(235..237,238..240,241..951)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(280..301)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(541..560)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     888..909
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             997..1017
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1030..1074
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1778..1796)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1792..1839
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1936..2391
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2023..2042)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2233..2254
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2417..2521
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2522..2590,2591..3382)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2740..2759)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3553..4141
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4042..4061
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4295..4312
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct
       61 agaaataatt ttgtttaact ttaagaagga gatatacata tgcggggttc tcatcatcat
      121 catcatcatg gtatggctag catgactggt ggacagcaaa tgggtcggga tctgtacgac
      181 gatgacgata aggatcgatg gggatccgag ctcgagatct gcagctggta ccagatggtg
      241 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
      301 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
      361 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
      421 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
      481 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
      541 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
      601 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
      661 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
      721 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
      781 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
      841 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
      901 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtccggaggg
      961 gatcggtgga gctccaccgg tggcggccgc tctagagaga atctttattt tcagggcgcc
     1021 gccaaattca aagaaaccgc tgctgctaaa ttcgaacgcc agcacatgga cagcggaggt
     1081 ggaggtacta gttctggagg cagcatgaag atggaggagc tgttcaagaa gcacaagatc
     1141 gtggccgtgc tgagagccaa cagcgtggag gaggccaaga agaaggccct ggccgtgttc
     1201 ctgggcggcg tgcacctgat cgagatcacc ttcaccgtgc ccgacgccga caccgtgatc
     1261 aaggagctga gcttcctgaa ggagatgggc gccatcatcg gcgccggcac cgtgaccagc
     1321 gtggagcagt gcagaaaggc cgtggagagc ggcgccgagt tcatcgtgag cccccacctg
     1381 gacgaggaga tcagccagtt ctgcaaggag aagggcgtgt tctacatgcc cggcgtgatg
     1441 acccccaccg agctggtgaa ggccatgaag ctgggccaca ccatcctgaa gctgttcccc
     1501 ggcgaggtgg tgggccccca gttcgtgaag gccatgaagg gccccttccc caacgtgaag
     1561 ttcgtgccca ccggcggcgt gaacctggac aacgtgtgcg agtggttcaa ggccggcgtg
     1621 ctggccgtgg gcgtgggcag cgccctggtg aagggcaccc ccgtggaggt ggccgagaag
     1681 gccaaggcct tcgtggagaa gatcagaggc tgcaccgagt gaaagcttga tccggctgct
     1741 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     1801 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     1861 ggatctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     1921 tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     1981 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     2041 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     2101 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     2161 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     2221 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     2281 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     2341 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     2401 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     2461 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     2521 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     2581 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     2641 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     2701 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     2761 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     2821 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     2881 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     2941 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     3001 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     3061 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     3121 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     3181 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     3241 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     3301 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     3361 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     3421 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     3481 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     3541 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     3601 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     3661 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     3721 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     3781 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     3841 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     3901 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     3961 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     4021 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     4081 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     4141 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     4201 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     4261 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     4321 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcag
//
