LOCUS       sequence-444670-        2375 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Medium copy cloning vector for integration of C-terminal FLAG 
            epitope tag. SmaI restriction site immediately upstream of the FLAG 
            epitope. FLAG epitope in same orientation as lac promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2375)
  AUTHORS   Braun HG, Kanwal N, Rivera Lopez LF, Thomassin JL
  TITLE     Generation of a plasmid series for rapid sub-cloning and use in 
            various Enterobacteriaceae.
  JOURNAL   J Biosci Bioeng. 2024 Sep 6:S1389-1723(24)00253-6. doi: 
            10.1016/j.jbiosc.2024.08.006.
  PUBMED    39244484
REFERENCE   2  (bases 1 to 2375)
  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: "J Biosci 
            Bioeng. 2024 Sep 6:S1389-1723(24)00253-6. doi: 
            10.1016/j.jbiosc.2024.08.006."
COMMENT     Sequence Label: pHBJT003
FEATURES             Location/Qualifiers
     source          1..2375
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(61..80)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      715..1260
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     1161..1180
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1377..1394
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1631..1652
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1667..1672,1673..1690,1691..1697)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1705..1721
                     /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     1710..1732
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1729..1745
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1729..1745
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1799..1822
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1858..1880)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        2267..2369
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             join(2370..2375,1..654)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
ORIGIN
        1 aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt
       61 gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg
      121 gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt
      181 cttgcccgcc tgatgaatgc tcatccggaa tttcgtatgg caatgaaaga cggtgagctg
      241 gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt
      301 tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa
      361 gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg
      421 tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat
      481 atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag
      541 gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc
      601 agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc gtaatttttt
      661 taaggcagtt attggtgcct agaaatattt tatctgatta ataagatgat cttcttgaga
      721 tcgttttggt ctgcgcgtaa tctcttgctc tgaaaacgaa aaaaccgcct tgcagggcgg
      781 tttttcgaag gttctctgag ctaccaactc tttgaaccga ggtaactggc ttggaggagc
      841 gcagtcacca aaacttgtcc tttcagttta gccttaaccg gcgcatgact tcaagactaa
      901 ctcctctaaa tcaattacca gtggctgctg ccagtggtgc ttttgcatgt ctttccgggt
      961 tggactcaag acgatagtta ccggataagg cgcagcggtc ggactgaacg gggggttcgt
     1021 gcatacagtc cagcttggag cgaactgcct acccggaact gagtgtcagg cgtggaatga
     1081 gacaaacgcg gccataacag cggaatgaca ccggtaaacc gaaaggcagg aacaggagag
     1141 cgcacgaggg agccgccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     1201 caccactgat ttgagcgtca gatttcgtga tgcttgtcag gggggcggag cctatggaaa
     1261 aacggctttg ccgcggccct ctcacttccc tgttaagtat cttcctggca tcttccagga
     1321 aatctccgcc ccgttcgtaa gccatttccg ctcgccgcag tcgaacgacc gagcgtagcg
     1381 agtcagtgag cgaggaagcg gaatatatcc tgtatcacat attctgctga cgcaccggtg
     1441 cagccttttt tctcctgcca catgaagcac ttcactgaca ccctcatcag tgccaacata
     1501 gtaagccagt atatacactc cgctagcgcc caatacgcaa accgcctctc cccgcgcgtt
     1561 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     1621 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc
     1681 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct
     1741 atgaccatga ttacgccaag cttgcatgcc tgcaggtcga ctctagagga tccccgggga
     1801 ctacaaagac gatgacgaca agtaatgata gggtaccgag ctcgaattca cggccgtcgt
     1861 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     1921 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     1981 gttgcgcagc ctgaatggcg aatggcgctg atgtccggcg gtgcttttgc cgttacgcac
     2041 caccccgtca gtagctgaac aggagggaca gctgatagaa acagaagcca ctggagcacc
     2101 tcaaaaacac catcatacac taaatcagta agttggcagc atcacccgac gcactttgcg
     2161 ccgaataaat acctgtgacg gaagatcact tcgcagaata aataaatcct ggtgtccctg
     2221 ttgataccgg gaagccctgg gccaactttt ggcgaaaatg agacgttgat cggcacgtaa
     2281 gaggttccaa ctttcaccat aatgaaataa gatcactacc gggcgtattt tttgagttat
     2341 cgagattttc aggagctaag gaagctaaaa tggag
//
