LOCUS       sequence-54327-e        6288 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PCR template for en passant (two-step Red-mediated) mutagenesis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6288)
  AUTHORS   Tischer BK, von Einem J, Kaufer B, Osterrieder N
  TITLE     Two-step red-mediated recombination for versatile high-efficiency 
            markerless DNA manipulation in Escherichia coli.
  JOURNAL   Biotechniques. 2006 Feb;40(2):191-7.
  PUBMED    16526409
REFERENCE   2  (bases 1 to 6288)
  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: 
            "Biotechniques."; date: "2006-02"; volume: "40(2)"; pages: "191-7"
COMMENT     Sequence Label: pEPkan-S
FEATURES             Location/Qualifiers
     source          1..6288
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             18..41
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(172..191)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             183..998
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             183..998
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(256..275)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     enhancer        1077..1456
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1457..1660
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1611..1631
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1705..1724
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1705..1723
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(1840..1858)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1841..1858)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1878..1895)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1884..2108
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2154..2582
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2241..2260)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2451..2472
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2591..2611)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2596..2926
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2777..2912
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2839..2858
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2993..3787
                     /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(3047..3066)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3657..3676
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3961..4082
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3998..4017)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4052..4071
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4131..4147)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4131..4147)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4144..4166)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4155..4171
                     /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(4179..4185,4186..4203,4204..4209))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4224..4245
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4362..4379)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4533..5121)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4613..4632)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5292..6083,6084..6152))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5915..5934
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6153..6257)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatccgat tacaaggatg acgacgataa gtagggataa cagggtaatc
       61 gatttattca acaaagccac gttgtgtctc aaaatctctg atgttacatt gcacaagata
      121 aaaatatatc atcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg
      181 ttatgagcca tattcaacgg gaaacgtctt gctcgaggcc gcgattaaat tccaacatgg
      241 atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa
      301 tctatcgatt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta
      361 gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc
      421 ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg
      481 cgatccccgg gaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata
      541 ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc
      601 cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt
      661 tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga
      721 aagaaatgca taagcttttg ccattctcac cggattcagt cgtcactcat ggtgatttct
      781 cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag
      841 tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt
      901 ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata
      961 aattgcagtt tcatttgatg ctcgatgagt ttttctaatc agaattggtt aattggttgt
     1021 aacactggca ttaccctgtt atccctagat cgatgtacgg gccagatata cgcgttgaca
     1081 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
     1141 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
     1201 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
     1261 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
     1321 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
     1381 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
     1441 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
     1501 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
     1561 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
     1621 cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc
     1681 cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc aagcttggta
     1741 ccgagctcgg atccactagt aacggccgcc agtgtgctgg aattctgcag atatccatca
     1801 cactggcggc cgctcgagca tgcatctaga gggccctatt ctatagtgtc acctaaatgc
     1861 tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     1921 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     1981 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     2041 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     2101 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
     2161 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     2221 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2281 cggctttccc cgtcaagctc taaatcgggg catcccttta gggttccgat ttagtgcttt
     2341 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     2401 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     2461 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     2521 tttggggatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     2581 ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccaggcagg
     2641 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     2701 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     2761 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     2821 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt
     2881 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc
     2941 ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga
     3001 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     3061 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     3121 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga
     3181 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     3241 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     3301 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     3361 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     3421 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     3481 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga
     3541 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     3601 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     3661 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     3721 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     3781 cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca
     3841 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     3901 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
     3961 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     4021 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     4081 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
     4141 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     4201 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     4261 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     4321 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     4381 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     4441 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     4501 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     4561 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     4621 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     4681 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcaatg ctcacgctgt
     4741 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     4801 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     4861 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     4921 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta
     4981 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     5041 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     5101 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     5161 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     5221 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     5281 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     5341 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     5401 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     5461 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     5521 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     5581 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     5641 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     5701 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     5761 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     5821 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     5881 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     5941 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     6001 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     6061 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     6121 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     6181 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     6241 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtc
//
