LOCUS       sequence-182505-        4741 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  yORF.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4741)
  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  JOURNAL   Unpublished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 4741)
  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            "Unpublished"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-A
FEATURES             Location/Qualifiers
     source          1..4741
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      147..735
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     636..655
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     889..906
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(987..1006)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1147..1169
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1163..1354)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     2812..2831
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2915..2934
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2915..2933
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2999..3018
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             3043..3065
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(3183..3201)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3197..3244
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3611..3616,3617..3633,3634..3639))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3666..3684)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3752..3856
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3857..3925,3926..4717)
                     /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(4075..4094)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
       61 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
      121 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
      181 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
      241 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
      301 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
      361 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
      421 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
      481 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
      541 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
      601 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
      661 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
      721 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
      781 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
      841 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
      901 agcgaggaag cggaagaggc gcgccatgcg gtattttctc cttacgcatc tgtgcggtat
      961 ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     1021 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
     1081 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     1141 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
     1201 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat
     1261 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg
     1321 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc
     1381 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg
     1441 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc
     1501 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag
     1561 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg
     1621 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg
     1681 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg
     1741 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga
     1801 gcacgatcat gcgcacccgt ggccaggacc caacgctgcc cgagatgcgc cgcgtgcggc
     1861 tgctggagat ggcggacgcg atggatatgt tctgccaagg gttggtttgc gcattcacag
     1921 ttctccgcaa gaattgattg gctccaattc ttggagtggt gaatccgtta gcgaggtgcc
     1981 gccggcttcc attcaggtcg aggtggcccg gctccatgca ccgcgacgca acgcggggag
     2041 gcagacaagg tatagggcgg cgcctacaat ccatgccaac ccgttccatg tgctcgccga
     2101 ggcggcataa atcgccgtga cgatcagcgg tccaatgatc gaagttaggc tggtaagagc
     2161 cgcgagcgat ccttgaagct gtccctgatg gtcgtcatct acctgcctgg acagcatggc
     2221 ctgcaacgcg ggcatcccga tgccgccgga agcgagaaga atcataatgg ggaaggccat
     2281 ccagcctcgc gtcgcgaacg ccagcaagac gtagcccagc gtgtcggccg ccatgccggc
     2341 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
     2401 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
     2461 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
     2521 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga
     2581 gctgactggg ttgaaggctc tcaagggcat cggtcgacgc tctcccttat gcgactcctg
     2641 cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg
     2701 tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac
     2761 gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc
     2821 ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc
     2881 ggcgtagagg atcgagatct cgatcccgcg aaattaatac gactcactat agggagacca
     2941 caacggtttc cctctagtgc cggctccgga gagctcttta atgcggccgc ttaattaagt
     3001 tcactgccgt ataggcagct aagaaaatta actagaaata attttgttta actttaagaa
     3061 ggagatatag atatcccaac tccataagga tcgcgatcgc cctgcaggcc ggccggtacc
     3121 acgcgtgcgc gctgatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
     3181 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     3241 tttgctgaaa ggaggaacta tatccggatt atccacagga cgggtgtggt cgccatgatc
     3301 gcgtagtcga tagtggctcc aagtagcgaa gcgagcagga ctgggcggcg gccaaagcgg
     3361 tcggacagtg ctccgagaac gggtgcgcat agaaattgca tcaacgcata tagcgctagc
     3421 agcacgccat agtgactggc gatgctgtcg gaatggacga aatcccgcaa gaggcccggc
     3481 agtaccggca taaccaagcc tatgcctaca gcatccaggg tgacggtgcc gaggatgacg
     3541 atgagcgcat tgttagattt catacacggt gcctgactgc gttagcaatt taactgtgat
     3601 aaactaccgc attaaagctt atcgatgata agctgtcaaa catgagaatt cttgaagacg
     3661 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     3721 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     3781 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataaca
     3841 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     3901 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     3961 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     4021 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     4081 tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     4141 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     4201 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     4261 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     4321 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     4381 gcgtgacacc acgatgcctg cagcaatggc aacaacgttg cgcaaactat taactggcga
     4441 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     4501 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     4561 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     4621 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     4681 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     4741 t
//
