LOCUS       sequence-444675-        4332 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Low copy cloning vector for integration of C-terminal FLAG epitope 
            tag (TetR). 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 4332)
  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 4332)
  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: pHBJT009
FEATURES             Location/Qualifiers
     source          1..4332
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             185..1135
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     promoter        join(1890..1895,1896..1912,1913..1918)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             1966..3156
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     primer_bind     complement(2318..2337)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3543..3565
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             complement(3601..3624)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(3678..3694)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3678..3694)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3691..3713)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3702..3718
                     /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)."
     promoter        complement(join(3726..3732,3733..3750,3751..3756))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3771..3792
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      join(4247..4332,1..137)
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
ORIGIN
        1 aaaaaaaaca cacaaaggtc tagcggaatt tacagagggt ctagcagaat ttacaagttt
       61 tccagcaaag gtctagcaga atttacagat acccacaact caaaggaaaa ggactagtaa
      121 ttatcattga ctagcccatc tcaattggta tagtgattaa aatcacctag accaattgag
      181 atgtatgtct gaattagttg ttttcaaagc aaatgaacta gcgattagtc gctatgactt
      241 aacggagcat gaaaccaagc taattttatg ctgtgtggca ctactcaacc ccacgattga
      301 aaaccctaca aggaaagaac ggacggtatc gttcacttat aaccaatacg ctcagatgat
      361 gaacatcagt agggaaaatg cttatggtgt attagctaaa gcaaccagag agctgatgac
      421 gagaactgtg gaaatcagga atcctttggt taaaggcttt gagattttcc agtggacaaa
      481 ctatgccaag ttctcaagcg aaaaattaga attagttttt agtgaagaga tattgcctta
      541 tcttttccag ttaaaaaaat tcataaaata taatctggaa catgttaagt cttttgaaaa
      601 caaatactct atgaggattt atgagtggtt attaaaagaa ctaacacaaa agaaaactca
      661 caaggcaaat atagagatta gccttgatga atttaagttc atgttaatgc ttgaaaataa
      721 ctaccatgag tttaaaaggc ttaaccaatg ggttttgaaa ccaataagta aagatttaaa
      781 cacttacagc aatatgaaat tggtggttga taagcgaggc cgcccgactg atacgttgat
      841 tttccaagtt gaactagata gacaaatgga tctcgtaacc gaacttgaga acaaccagat
      901 aaaaatgaat ggtgacaaaa taccaacaac cattacatca gattcctacc tacataacgg
      961 actaagaaaa acactacacg atgctttaac tgcaaaaatt cagctcacca gttttgaggc
     1021 aaaatttttg agtgacatgc aaagtaagta tgatctcaat ggttcgttct catggctcac
     1081 gcaaaaacaa cgaaccacac tagagaacat actggctaaa tacggaagga tctgaggttc
     1141 ttatggctct tgtatctatc agtgaagcat caagactaac aaacaaaagt agaacaactg
     1201 ttcaccgtta catatcaaag ggaaaactgt ccatatgcac agatgaaaac ggtgtaaaaa
     1261 agatagatac atcagagctt ttacgagttt ttggtgcatt caaagctgtt caccatgaac
     1321 agatcgacaa tgtaacagat gaacagcatg taacacctaa tagaacaggt gaaaccagta
     1381 aaacaaagca actagaacat gaaattgaac acctgagaca acttgttaca gctcaacagt
     1441 cacacataga cagcctgaaa caggcgatgc tgcttatcga atcaaagctg ccgacaacac
     1501 gggagccagt gacgcctccc gtggggaaaa aatcatggca attctggaag aaatagcgct
     1561 ttcagccggc aaaccggctg aagccggatc tgcgattctg ataacaaact agcaacacca
     1621 gaacagcccg tttgcgggca gcaaaacccg tacttttgga cgttccggcg gttttttgtg
     1681 gcgagtggtg ttcgggcggt gcgcgcaaga tccattatgt taaacgggcg agtttacatc
     1741 tcaaaaccgc ccgcttaaca ccatcagaaa tcctcagcgc gattttaagc accaaccccc
     1801 ccccgtaaca cccaaatcca tactgaaagt ggctttgttg aataaatcag atttcgggta
     1861 agtctcctag gcaccaataa ctgccatgtt tgacagctta tcatcgataa gctttaatgc
     1921 ggtagtttat cacagttaaa ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat
     1981 gcgctcatcg tcatcctcgg caccgtcacc ctggatgctg taggcatagg cttggttatg
     2041 ccggtactgc cgggcctctt gcgggatatc gtccattccg acagcatcgc cagtcactat
     2101 ggcgtgctgc tagcgctata tgcgttgatg caatttctat gcgcacccgt tctcggagca
     2161 ctgtccgacc gctttggccg ccgcccagtc ctgctcgctt cgctacttgg agccactatc
     2221 gactacgcga tcatggcgac cacacccgtc ctgtggatcc tctacgccgg acgcatcgtg
     2281 gccggcatca ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat
     2341 ggggaagatc gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg
     2401 gcaggccccg tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg
     2461 gcggcggtgc tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat
     2521 aagggagagc gtcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg
     2581 gcgcggggca tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta
     2641 ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg
     2701 acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc tcaagccttc
     2761 gtcactggtc ccgccaccaa acgtttcggc gagaagcagg ccattatcgc cggcatggcg
     2821 gccgacgcgc tgggctacgt cttgctggcg ttcgcgacgc gaggctggat ggccttcccc
     2881 attatgattc ttctcgcttc cggcggcatc gggatgcccg cgttgcaggc catgctgtcc
     2941 aggcaggtag atgacgacca tcagggacag cttcaaggat cgctcgcggc tcttaccagc
     3001 ctaacttcga tcattggacc gctgatcgtc acggcgattt atgccgcctc ggcgagcaca
     3061 tggaacgggt tggcatggat tgtaggcgcc gccctatacc ttgtctgcct ccccgcgttg
     3121 cgtcgcggtg catggagccg ggccacctcg acctgaacgt ctcattttcg ccaaaagttg
     3181 gcccagggct tcccggtatc aacagggaca ccaggattta tttattctgc gaagtgatct
     3241 tccgtcacag gtatttattc ggcgcaaagt gcgtcgggtg atgctgccaa cttactgatt
     3301 tagtgtatga tggtgttttt gaggtgctcc agtggcttct gtttctatca gctgtccctc
     3361 ctgttcagct actgacgggg tggtgcgtaa cggcaaaagc accgccggac atcagcgcca
     3421 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
     3481 acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt
     3541 ttcccagtca cgacgttgta aaacgacggc cgtgaattcg agctcggtac cctatcatta
     3601 cttgtcgtca tcgtctttgt agtccccggg gatcctctag agtcgacctg caggcatgca
     3661 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     3721 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     3781 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     3841 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctcc
     3901 tcgctaacgg attcaccact ccgcagaccc gccataaaac gccctgagaa gcccgtgacg
     3961 ggcttttctt gtattatggg tagtttcctt gcatgaatcc ataaaaggcg cctgtagtgc
     4021 catttacccc cattcactgc cagagccgtg agcgcagcga actgaatgtc acgaaaaaga
     4081 cagcgactca ggtgcctgat ggtcggagac aaaaggaata ttcagcgatt tgcccgagct
     4141 tgcgagggtg ctacttaagc ctttagggtt ttaaggtctg ttttgtagag gagcaaacag
     4201 cgtttgcgac atccttttgt aatactgcgg aactgactaa agtagtgagt tatacacagg
     4261 gctgggatct attcttttta tcttttttta ttctttcttt attctataaa ttataaccac
     4321 ttgaatataa ac
//
