LOCUS       sequence-444678-        3698 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Low copy cloning vector for integration of C-terminal FLAG epitope 
            tag (GmR). SmaI restriction site immediately upstream of the FLAG 
            epitope. FLAG epitope in opposite orientation of lac promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3698)
  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 3698)
  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: pHBJT012
FEATURES             Location/Qualifiers
     source          1..3698
                     /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"
     CDS             complement(1886..2419)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(2420..2522)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     2909..2931
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2923..2940
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2924..2940
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2985..3008
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(3044..3060)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3044..3060)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3057..3079)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3068..3084
                     /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(3092..3098,3099..3116,3117..3122))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3137..3158
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      join(3613..3698,1..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 ctgccttagg tggcggtact tgggtcgata tcaaagtgca
     1921 tcacttcttc ccgtatgccc aactttgtat agagagccac tgcgggatcg tcaccgtaat
     1981 ctgcttgcac gtagatcaca taagcaccaa gcgcgttggc ctcatgcttg aggagattga
     2041 tgagcgcggt ggcaatgccc tgcctccggt gctcgccgga gactgcgaga tcatagatat
     2101 agatctcact acgcggctgc tcaaacttgg gcagaacgta agccgcgaga gcgccaacaa
     2161 ccgcttcttg gtcgaaggca gcaagcgcga tgaatgtctt actacggagc aagttcccga
     2221 ggtaatcgga gtccggctga tgttgggagt aggtggctac gtctccgaac tcacgaccga
     2281 aaagatcaag agcagcccgc atggatttga cttggtcagg gccgagccta catgtgcgaa
     2341 tgatgcccat acttgagcca cctaactttg ttttagggcg actgccctgc tgcgtaacat
     2401 cgttgctgct gcgtaacatt ttagcttcct tagctcctga aaatctcgat aactcaaaaa
     2461 atacgcccgg tagtgatctt atttcattat ggtgaaagtt ggaacctctt acgtgccgat
     2521 caacgtctca ttttcgccaa aagttggccc agggcttccc ggtatcaaca gggacaccag
     2581 gatttattta ttctgcgaag tgatcttccg tcacaggtat ttattcggcg caaagtgcgt
     2641 cgggtgatgc tgccaactta ctgatttagt gtatgatggt gtttttgagg tgctccagtg
     2701 gcttctgttt ctatcagctg tccctcctgt tcagctactg acggggtggt gcgtaacggc
     2761 aaaagcaccg ccggacatca gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg
     2821 cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg
     2881 cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt
     2941 gccaagcttg catgcctgca ggtcgactct agaggatccc cggggactac aaagacgatg
     3001 acgacaagta atgatagggt accgagctcg aattcgtaat catgtcatag ctgtttcctg
     3061 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     3121 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     3181 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     3241 gaggcggttt gcgtattggg cgctcctcgc taacggattc accactccgc agacccgcca
     3301 taaaacgccc tgagaagccc gtgacgggct tttcttgtat tatgggtagt ttccttgcat
     3361 gaatccataa aaggcgcctg tagtgccatt tacccccatt cactgccaga gccgtgagcg
     3421 cagcgaactg aatgtcacga aaaagacagc gactcaggtg cctgatggtc ggagacaaaa
     3481 ggaatattca gcgatttgcc cgagcttgcg agggtgctac ttaagccttt agggttttaa
     3541 ggtctgtttt gtagaggagc aaacagcgtt tgcgacatcc ttttgtaata ctgcggaact
     3601 gactaaagta gtgagttata cacagggctg ggatctattc tttttatctt tttttattct
     3661 ttctttattc tataaattat aaccacttga atataaac
//
