LOCUS       sequence-347003-        6155 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A template plasmid for amplification of the cI-hok-neo cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6155)
  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 6155)
  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: pTmpKm
FEATURES             Location/Qualifiers
     source          1..6155
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             45..56
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             complement(232..1026)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(343..362)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     953..972
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(1170..1186)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1170..1186)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1183..1205)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1194..1210
                     /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(1218..1224,1225..1242,1243..1248))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1263..1284
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(join(1961..2752,2753..2821))
                     /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     2584..2603
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2822..2926)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2994..3012
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      3511..3733
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             3781..4731
                     /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     5280..5302
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5294..5311
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5295..5311
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(5395..6108)
                     /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 ggagcggcga taccgtaaag
      301 cacgaggaag cggtcagccc attcgccgcc aagctcttca gcaatatcac gggtagccaa
      361 cgctatgtcc tgatagcggt ccgccacacc cagccggcca cagtcgatga atccagaaaa
      421 gcggccattt tccaccatga tattcggcaa gcaggcatcg ccatgggtca cgacgagatc
      481 ctcgccgtcg ggcatgcgcg ccttgagcct ggcgaacagt tcggctggcg cgagcccctg
      541 atgctcttcg tccagatcat cctgatcgac aagaccggct tccatccgag tacgtgctcg
      601 ctcgatgcga tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa gcgtatgcag
      661 ccgccgcatt gcatcagcca tgatggatac tttctcggca ggagcaaggt gagatgacag
      721 gagatcctgc cccggcactt cgcccaatag cagccagtcc cttcccgctt cagtgacaac
      781 gtcgagcaca gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc gcgctgcctc
      841 gtcctgcagt tcattcaggg caccggacag gtcggtcttg acaaaaagaa ccgggcgccc
      901 ctgcgctgac agccggaaca cggcggcatc agagcagccg attgtctgtt gtgcccagtc
      961 atagccgaat agcctctcca cccaagcggc cggagaacct gcgtgcaatc catcttgttc
     1021 aatcatgcga aacgatcctc atcctgtatg gtaccattat aaccgccaag cttaaaatgt
     1081 caacattttt aataccttta agtgatacca gatggcattg gctcacaatt ccacacaaca
     1141 tgggtaccga gctcgaattc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     1201 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     1261 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     1321 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     1381 attgggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     1441 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     1501 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     1561 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     1621 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     1681 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     1741 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     1801 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     1861 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     1921 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     1981 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     2041 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     2101 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     2161 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     2221 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctgcag
     2281 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     2341 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     2401 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     2461 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     2521 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaacac
     2581 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     2641 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     2701 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     2761 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     2821 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     2881 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     2941 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     3001 gtatcacgag gccctttcgt cttcaagaat tgacagtaag acgggtaagc ctgttgatga
     3061 taccgctgcc ttactgggtg cattagccag tctgaatgac ctgtcacggg ataatccgaa
     3121 gtggtcagac tggaaaatca gagggcagga actgctgaac agcaaaaagt cagatagcac
     3181 cacatagcag acccgccata aaacgccctg agaagcccgt gacgggcttt tcttgtatta
     3241 tgggtagttt ccttgcatga atccataaaa ggcgcctgta gtgccattta cccccattca
     3301 ctgccagagc cgtgagcgca gcgaactgaa tgtcacgaaa aagacagcga ctcaggtgcc
     3361 tgatggtcgg agacaaaagg aatattcagc gatttgcccg agcttgcgag ggtgctactt
     3421 aagcctttag ggttttaagg tctgttttgt agaggagcaa acagcgtttg cgacatcctt
     3481 ttgtaatact gcggaactga ctaaagtagt gagttataca cagggctggg atctattctt
     3541 tttatctttt tttattcttt ctttattcta taaattataa ccacttgaat ataaacaaaa
     3601 aaaacacaca aaggtctagc ggaatttaca gagggtctag cagaatttac aagttttcca
     3661 gcaaaggtct agcagaattt acagataccc acaactcaaa ggaaaaggac tagtaattat
     3721 cattgactag cccatctcaa ttggtatagt gattaaaatc acctagacca attgagatgt
     3781 atgtctgaat tagttgtttt caaagcaaat gaactagcga ttagtcgcta tgacttaacg
     3841 gagcatgaaa ccaagctaat tttatgctgt gtggcactac tcaaccccac gattgaaaac
     3901 cctacaagga aagaacggac ggtatcgttc acttataacc aatacgctca gatgatgaac
     3961 atcagtaggg aaaatgctta tggtgtatta gctaaagcaa ccagagagct gatgacgaga
     4021 actgtggaaa tcaggaatcc tttggttaaa ggctttgaga ttttccagtg gacaaactat
     4081 gccaagttct caagcgaaaa attagaatta gtttttagtg aagagatatt gccttatctt
     4141 ttccagttaa aaaaattcat aaaatataat ctggaacatg ttaagtcttt tgaaaacaaa
     4201 tactctatga ggatttatga gtggttatta aaagaactaa cacaaaagaa aactcacaag
     4261 gcaaatatag agattagcct tgatgaattt aagttcatgt taatgcttga aaataactac
     4321 catgagttta aaaggcttaa ccaatgggtt ttgaaaccaa taagtaaaga tttaaacact
     4381 tacagcaata tgaaattggt ggttgataag cgaggccgcc cgactgatac gttgattttc
     4441 caagttgaac tagatagaca aatggatctc gtaaccgaac ttgagaacaa ccagataaaa
     4501 atgaatggtg acaaaatacc aacaaccatt acatcagatt cctacctaca taacggacta
     4561 agaaaaacac tacacgatgc tttaactgca aaaattcagc tcaccagttt tgaggcaaaa
     4621 tttttgagtg acatgcaaag taagtatgat ctcaatggtt cgttctcatg gctcacgcaa
     4681 aaacaacgaa ccacactaga gaacatactg gctaaatacg gaaggatctg aggttcttat
     4741 ggctcttgta tctatcagtg aagcatcaag actaacaaac aaaagtagaa caactgttca
     4801 ccgttacata tcaaagggaa aactgtccat atgcacagat gaaaacggtg taaaaaagat
     4861 agatacatca gagcttttac gagtttttgg tgcattcaaa gctgttcacc atgaacagat
     4921 cgacaatgta acagatgaac agcatgtaac acctaataga acaggtgaaa ccagtaaaac
     4981 aaagcaacta gaacatgaaa ttgaacacct gagacaactt gttacagctc aacagtcaca
     5041 catagacagc ctgaaacagg cgatgctgct tatcgaatca aagctgccga caacacggga
     5101 gccagtgacg cctcccgtgg ggaaaaaatc atggcaattc tggaagaaat agcgccattc
     5161 gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg
     5221 ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc
     5281 ccagtcacga cgttgtaaaa cgacggccag tgccaagctt tttcgaaaaa aggcctccca
     5341 aatcgggggg ccttttttat tgataacaaa acgccgacca gaacaccttg ccgatcagcc
     5401 aaacgtctct tcaggccact gactagcgat aactttcccc acaacggaac aactctcatt
     5461 gcatgggatc attgggtact gtgggtttag tggttgtaaa aacacctgac cgctatccct
     5521 gatcagtttc ttgaaggtaa actcatcacc cccaagtctg gctatgcaga aatcacctgg
     5581 ctcaacagcc tgctcagggt caacgagaat taacattccg tcaggaaagc ttggcttgga
     5641 gcctgttggt gcggtcatgg aattaccttc aacctcaagc cagaatgcag aatcactggc
     5701 ttttttggtt gtgcttaccc atctctccgc atcacctttg gtaaaggttc taagcttagg
     5761 tgagaacatc cctgcctgaa catgagaaaa aacagggtac tcatactcac ttctaagtga
     5821 cggctgcata ctaaccgctt catacatctc gtagatttct ctggcgattg aagggctaaa
     5881 ttcttcaacg ctaactttga gaatttttgc aagcaatgcg gcgttataag catttaatgc
     5941 attgatgcca ttaaataaag caccaacgcc tgactgcccc atccccatct tgtctgcgac
     6001 agattcctgg gataagccaa gttcattttt ctttttttca taaattgctt taaggcgacg
     6061 tgcgtcctca agctgctctt gtgttaatgg tttctttttt gtgctcatac gttaaatcta
     6121 tcaccgcaag ggataaatat ctaacaccgt gcgtg
//
