LOCUS       sequence-182506-        5527 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  His6-GFP-tev-yORF.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5527)
  TITLE     Series-371: N- or C-terminal Tag E.coli T7 Expression Vectors with 
            Biobrick Polycistronic Restriction Sites and Csy4 cleavage
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5527)
  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: 
            "Unpublished"
COMMENT     Sequence Label: 371-GFP
FEATURES             Location/Qualifiers
     source          1..5527
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(18..36)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        104..208
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(209..277,278..1069)
                     /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(427..446)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1240..1828
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1729..1748
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1982..1999
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2080..2099)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2240..2262
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2256..2447)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     3905..3924
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4008..4027
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4008..4026
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4092..4111
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             4136..4158
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4178..4195
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(4205..4207,4208..4918)
                     /codon_start=1
                     /product="enhanced GFP (EGFP) derivative that exhibits 
                     robust folding and enhanced photostability (Zhong et al., 
                     2019)"
                     /label=SiriusGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(4247..4268)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     4855..4876
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4928..4948
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(5063..5081)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5077..5124
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(5490..5495,5496..5512,5513..5518))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
ORIGIN
        1 ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
       61 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
      121 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
      181 gcttcaataa cattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
      241 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
      301 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
      361 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
      421 agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg
      481 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
      541 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
      601 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
      661 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
      721 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact
      781 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
      841 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
      901 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
      961 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     1021 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     1081 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     1141 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     1201 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     1261 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     1321 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     1381 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     1441 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     1501 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     1561 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     1621 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     1681 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     1741 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     1801 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     1861 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     1921 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     1981 cagcgagtca gtgagcgagg aagcggaaga ggcgcgccat gcggtatttt ctccttacgc
     2041 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
     2101 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     2161 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     2221 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     2281 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     2341 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     2401 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     2461 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     2521 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     2581 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     2641 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     2701 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     2761 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     2821 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     2881 ctcaacgaca ggagcacgat catgcgcacc cgtggccagg acccaacgct gcccgagatg
     2941 cgccgcgtgc ggctgctgga gatggcggac gcgatggata tgttctgcca agggttggtt
     3001 tgcgcattca cagttctccg caagaattga ttggctccaa ttcttggagt ggtgaatccg
     3061 ttagcgaggt gccgccggct tccattcagg tcgaggtggc ccggctccat gcaccgcgac
     3121 gcaacgcggg gaggcagaca aggtataggg cggcgcctac aatccatgcc aacccgttcc
     3181 atgtgctcgc cgaggcggca taaatcgccg tgacgatcag cggtccaatg atcgaagtta
     3241 ggctggtaag agccgcgagc gatccttgaa gctgtccctg atggtcgtca tctacctgcc
     3301 tggacagcat ggcctgcaac gcgggcatcc cgatgccgcc ggaagcgaga agaatcataa
     3361 tggggaaggc catccagcct cgcgtcgcga acgccagcaa gacgtagccc agcgcgtcgg
     3421 ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga
     3481 cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg
     3541 tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc
     3601 ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg
     3661 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga cgctctccct
     3721 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     3781 ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg
     3841 ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc
     3901 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
     3961 ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa tacgactcac
     4021 tatagggaga ccacaacggt ttccctctag tgccggctcc ggagagctct ttaatgcggc
     4081 cgcttaatta agttcactgc cgtataggca gctaagaaaa ttaactagaa ataattttgt
     4141 ttaactttaa gaaggagata tacatatgaa atcttctcac catcaccatc accatggttc
     4201 ttctgtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     4261 cggcgacgta aacggccaca agttcagcgt gcgcggcgag ggcgagggcg atgccaccaa
     4321 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     4381 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     4441 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctc
     4501 cttcaaggac gacggcacct acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     4561 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     4621 caagctggag tacaacttca acagccacaa cgtctatatc acggccgaca agcagaagaa
     4681 cggcatcaag gcgaacttca agatccgcca caacgtcgag gacggcagcg tgcagctcgc
     4741 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     4801 ctacctgagc acccagtcca agctgagcaa agaccccaac gagaagcgcg atcacatggt
     4861 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaaggg
     4921 gatcgaggaa aacctgtact tccaatccaa tattggaagt ggataacgga tcgcgatcgc
     4981 cctgcaggcc ggccggtacc acgcgtgcgc gctgatccgg ctgctaacaa agcccgaaag
     5041 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     5101 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggata tccacaggac
     5161 gggtgtggtc gccatgatcg cgtagtcgat agtggctcca agtagcgaag cgagcaggac
     5221 tgggcggcgg ccaaagcggt cggacagtgc tccgagaacg ggtgcgcata gaaattgcat
     5281 caacgcatat agcgctagca gcacgccata gtgactggcg atgctgtcgg aatggacgat
     5341 atcccgcaag aggcccggca gtaccggcat aaccaagcct atgcctacag catccagggt
     5401 gacggtgccg aggatgacga tgagcgcatt gttagatttc atacacggtg cctgactgcg
     5461 ttagcaattt aactgtgata aactaccgca ttaaagctta tcgatgataa gctgtcaaac
     5521 atgagaa
//
