LOCUS       sequence-347009-        6219 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A template plasmid for amplification of the cI-hok-aacC4 cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6219)
  AUTHORS   Bubnov DM, Yuzbashev TV, Khozov AA, Melkina OE, Vybornaya TV, Stan 
            GB, Sineoky SP
  TITLE     Robust counterselection and advanced lambdaRed recombineering enable
            markerless chromosomal integration of large heterologous constructs.
  JOURNAL   Nucleic Acids Res. 2022 Aug 26;50(15):8947-8960. doi: 
            10.1093/nar/gkac649.
  PUBMED    35920321
REFERENCE   2  (bases 1 to 6219)
  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"; doi: "10"; journalName: 
            "Nucleic Acids Res"; date: "2022-08-26- 26"; volume: "50"; issue: 
            "15"; pages: "8947-8960"
COMMENT     Sequence Label: pTmpGm
FEATURES             Location/Qualifiers
     source          1..6219
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             45..56
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             complement(285..1085)
                     /codon_start=1
                     /gene="aac(3)IV"
                     /product="aminoglycoside 3-N-acetyltransferase type IV     
                               "
                     /label=ApmR
                     /note="confers resistance to apramycin (Paget and Davies, 
                     1996)"
                     /translation="MSSAVECNVVQYEWRKAELIGQLLNLGVTPGGVLLVHSSFRSVRP
                     LEDGPLGLIEALRAALGPGGTLVMPSWSGLDDEPFDPATSPVTPDLGVVSDTFWRLPNV
                     KRSAHPFAFAAAGPQAEQIISDPLPLPPHSPASPVARVHELDGQVLLLGVGHDANTTLH
                     LAELMAKVPYGVPRHCTILQDGKLVRVDYLENDHCCERFALADRWLKEKSLQKEGPVGH
                     AFARLIRSRDIVATALGQLGRDPLIFLHPPEAGCEECDAARQSIG"
     primer_bind     complement(1234..1250)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1234..1250)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1247..1269)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1258..1274
                     /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(1282..1288,1289..1306,1307..1312))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1327..1348
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(join(2025..2816,2817..2885))
                     /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     2648..2667
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2886..2990)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3058..3076
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      3575..3797
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             3845..4795
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     primer_bind     5344..5366
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5358..5375
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5359..5375
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(5459..6172)
                     /codon_start=1
                     /gene="cI"
                     /product="phage lambda repressor"
                     /label=lambda repressor
                     /translation="MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQ
                     SGVGALFNGINALNAYNAALLAKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY
                     PVFSHVQAGMFSPKLRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
                     GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV
                     VGKVIASQWPEETFG"
ORIGIN
        1 ttgactattt tacctctggc ggtgataatg gttgcatgta ctttaaagag gagaaattaa
       61 ctatgaaact accacgaagt tcccttgtct ggtgtgtgtt gatcgtgtgt ctcacactgt
      121 tgatattcac ttatctgaca cgaaaatcgc tgtgcgagat tcgttacaga gacggacaca
      181 gggaggtggc ggctttcatg gcttacgaat ccggtaagta gcaacctgga gttagaagaa
      241 ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg gtcagccaat cgactggcga
      301 gcggcatcgc attcttcgca tcccgcctct ggcggatgca ggaagatcaa cggatctcgg
      361 cccagttgac ccagggctgt cgccacaatg tcgcgggagc ggatcaaccg agcaaaggca
      421 tgaccgactg gaccttcctt ctgaaggctc ttctccttga gccacctgtc cgccaaggca
      481 aagcgctcac agcagtggtc attctcgaga taatcgacgc gtaccaactt gccatcctga
      541 agaatggtgc agtgtctcgg caccccatag ggaacctttg ccatcaactc ggcaagatgc
      601 agcgtcgtgt tggcatcgtg tcccacgccg aggagaagta cctgcccatc gagttcatgg
      661 acacgggcga ccgggcttgc aggcgagtga ggtggcaggg gcaatggatc agagatgatc
      721 tgctctgcct gtggccccgc tgccgcaaag gcaaatggat gggcgctgcg ctttacattt
      781 ggcaggcgcc agaatgtgtc agagacaact ccaaggtccg gtgtaacggg cgacgtggca
      841 ggatcgaacg gctcgtcgtc cagacctgac cacgagggca tgacgagcgt ccctcccgga
      901 cccagcgcag cacgcagggc ctcgatcagt ccaagtggcc catcttcgag gggccggacg
      961 ctacggaagg agctgtggac cagcagcaca ccgccggggg taaccccaag gttgagaagc
     1021 tgaccgatga gctcggcttt tcgccattcg tattgcacga cattgcactc caccgctgat
     1081 gacatatgta tatctccttc tctcatcctg tatggtacca ttataaccgc caagcttaaa
     1141 atgtcaacat ttttaatacc tttaagtgat accagatggc attggctcac aattccacac
     1201 aacatgggta ccgagctcga attcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     1261 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     1321 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     1381 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     1441 gcgtattggg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     1501 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     1561 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     1621 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     1681 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca
     1741 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     1801 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     1861 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     1921 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     1981 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     2041 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     2101 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     2161 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     2221 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     2281 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     2341 gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     2401 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     2461 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     2521 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     2581 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     2641 acacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     2701 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     2761 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     2821 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     2881 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     2941 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     3001 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
     3061 aggcgtatca cgaggccctt tcgtcttcaa gaattgacag taagacgggt aagcctgttg
     3121 atgataccgc tgccttactg ggtgcattag ccagtctgaa tgacctgtca cgggataatc
     3181 cgaagtggtc agactggaaa atcagagggc aggaactgct gaacagcaaa aagtcagata
     3241 gcaccacata gcagacccgc cataaaacgc cctgagaagc ccgtgacggg cttttcttgt
     3301 attatgggta gtttccttgc atgaatccat aaaaggcgcc tgtagtgcca tttaccccca
     3361 ttcactgcca gagccgtgag cgcagcgaac tgaatgtcac gaaaaagaca gcgactcagg
     3421 tgcctgatgg tcggagacaa aaggaatatt cagcgatttg cccgagcttg cgagggtgct
     3481 acttaagcct ttagggtttt aaggtctgtt ttgtagagga gcaaacagcg tttgcgacat
     3541 ccttttgtaa tactgcggaa ctgactaaag tagtgagtta tacacagggc tgggatctat
     3601 tctttttatc tttttttatt ctttctttat tctataaatt ataaccactt gaatataaac
     3661 aaaaaaaaca cacaaaggtc tagcggaatt tacagagggt ctagcagaat ttacaagttt
     3721 tccagcaaag gtctagcaga atttacagat acccacaact caaaggaaaa ggactagtaa
     3781 ttatcattga ctagcccatc tcaattggta tagtgattaa aatcacctag accaattgag
     3841 atgtatgtct gaattagttg ttttcaaagc aaatgaacta gcgattagtc gctatgactt
     3901 aacggagcat gaaaccaagc taattttatg ctgtgtggca ctactcaacc ccacgattga
     3961 aaaccctaca aggaaagaac ggacggtatc gttcacttat aaccaatacg ctcagatgat
     4021 gaacatcagt agggaaaatg cttatggtgt attagctaaa gcaaccagag agctgatgac
     4081 gagaactgtg gaaatcagga atcctttggt taaaggcttt gagattttcc agtggacaaa
     4141 ctatgccaag ttctcaagcg aaaaattaga attagttttt agtgaagaga tattgcctta
     4201 tcttttccag ttaaaaaaat tcataaaata taatctggaa catgttaagt cttttgaaaa
     4261 caaatactct atgaggattt atgagtggtt attaaaagaa ctaacacaaa agaaaactca
     4321 caaggcaaat atagagatta gccttgatga atttaagttc atgttaatgc ttgaaaataa
     4381 ctaccatgag tttaaaaggc ttaaccaatg ggttttgaaa ccaataagta aagatttaaa
     4441 cacttacagc aatatgaaat tggtggttga taagcgaggc cgcccgactg atacgttgat
     4501 tttccaagtt gaactagata gacaaatgga tctcgtaacc gaacttgaga acaaccagat
     4561 aaaaatgaat ggtgacaaaa taccaacaac cattacatca gattcctacc tacataacgg
     4621 actaagaaaa acactacacg atgctttaac tgcaaaaatt cagctcacca gttttgaggc
     4681 aaaatttttg agtgacatgc aaagtaagta tgatctcaat ggttcgttct catggctcac
     4741 gcaaaaacaa cgaaccacac tagagaacat actggctaaa tacggaagga tctgaggttc
     4801 ttatggctct tgtatctatc agtgaagcat caagactaac aaacaaaagt agaacaactg
     4861 ttcaccgtta catatcaaag ggaaaactgt ccatatgcac agatgaaaac ggtgtaaaaa
     4921 agatagatac atcagagctt ttacgagttt ttggtgcatt caaagctgtt caccatgaac
     4981 agatcgacaa tgtaacagat gaacagcatg taacacctaa tagaacaggt gaaaccagta
     5041 aaacaaagca actagaacat gaaattgaac acctgagaca acttgttaca gctcaacagt
     5101 cacacataga cagcctgaaa caggcgatgc tgcttatcga atcaaagctg ccgacaacac
     5161 gggagccagt gacgcctccc gtggggaaaa aatcatggca attctggaag aaatagcgcc
     5221 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
     5281 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
     5341 tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gctttttcga aaaaaggcct
     5401 cccaaatcgg ggggcctttt ttattgataa caaaacgccg accagaacac cttgccgatc
     5461 agccaaacgt ctcttcaggc cactgactag cgataacttt ccccacaacg gaacaactct
     5521 cattgcatgg gatcattggg tactgtgggt ttagtggttg taaaaacacc tgaccgctat
     5581 ccctgatcag tttcttgaag gtaaactcat cacccccaag tctggctatg cagaaatcac
     5641 ctggctcaac agcctgctca gggtcaacga gaattaacat tccgtcagga aagcttggct
     5701 tggagcctgt tggtgcggtc atggaattac cttcaacctc aagccagaat gcagaatcac
     5761 tggctttttt ggttgtgctt acccatctct ccgcatcacc tttggtaaag gttctaagct
     5821 taggtgagaa catccctgcc tgaacatgag aaaaaacagg gtactcatac tcacttctaa
     5881 gtgacggctg catactaacc gcttcataca tctcgtagat ttctctggcg attgaagggc
     5941 taaattcttc aacgctaact ttgagaattt ttgcaagcaa tgcggcgtta taagcattta
     6001 atgcattgat gccattaaat aaagcaccaa cgcctgactg ccccatcccc atcttgtctg
     6061 cgacagattc ctgggataag ccaagttcat ttttcttttt ttcataaatt gctttaaggc
     6121 gacgtgcgtc ctcaagctgc tcttgtgtta atggtttctt ttttgtgctc atacgttaaa
     6181 tctatcaccg caagggataa atatctaaca ccgtgcgtg
//
