LOCUS       sequence-100412-        6441 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 6441)
  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 6441)
  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-S2
FEATURES             Location/Qualifiers
     source          1..6441
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             913..936
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1067..1086)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1078..1893
                     /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             1078..1893
                     /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(1151..1170)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1993..2011)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1994..2011)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2031..2048)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2037..2261
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2307..2735
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2394..2413)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2604..2625
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2744..2764)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2749..3079
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2930..3065
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2992..3011
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3146..3940
                     /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(3200..3219)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3810..3829
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4114..4235
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4151..4170)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4205..4224
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4284..4300)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4284..4300)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4297..4319)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4308..4324
                     /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(4332..4338,4339..4356,4357..4362))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4377..4398
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4515..4532)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4686..5274)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4766..4785)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5445..6236,6237..6305))
                     /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     6068..6087
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6306..6410)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 gagctcggat ccgattacaa ggatgacgac gataagtagg gataacaggg taatcgattt
      961 attcaacaaa gccacgttgt gtctcaaaat ctctgatgtt acattgcaca agataaaaat
     1021 atatcatcat gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg
     1081 agccatattc aacgggaaac gtcttgctcg aggccgcgat taaattccaa catggatgct
     1141 gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat
     1201 cgattgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt
     1261 gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt
     1321 ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc
     1381 cccgggaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt
     1441 gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt
     1501 aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt
     1561 gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa
     1621 atgcataagc ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt
     1681 gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga
     1741 atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct
     1801 tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg
     1861 cagtttcatt tgatgctcga tgagtttttc taatcagaat tggttaattg gttgtaacac
     1921 tggcattacc ctgttatccc tagatatcca tcacactggc ggccgctcga gcatgcatct
     1981 agagggccct attctatagt gtcacctaaa tgctagagct cgctgatcag cctcgactgt
     2041 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     2101 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     2161 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     2221 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     2281 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     2341 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     2401 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     2461 gggcatccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     2521 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     2581 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     2641 tatctcggtc tattcttttg atttataagg gattttgggg atttcggcct attggttaaa
     2701 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
     2761 gggtgtggaa agtccccagg ctccccaggc aggcagaagt atgcaaagca tgcatctcaa
     2821 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     2881 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     2941 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     3001 agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg
     3061 aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca
     3121 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     3181 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     3241 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     3301 cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac
     3361 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     3421 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     3481 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     3541 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     3601 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     3661 caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     3721 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     3781 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     3841 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     3901 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa
     3961 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     4021 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     4081 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
     4141 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     4201 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     4261 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     4321 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     4381 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     4441 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     4501 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     4561 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     4621 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     4681 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     4741 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     4801 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     4861 ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     4921 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     4981 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     5041 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     5101 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca
     5161 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     5221 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     5281 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     5341 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     5401 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     5461 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     5521 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     5581 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     5641 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     5701 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     5761 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     5821 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     5881 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     5941 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     6001 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     6061 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     6121 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     6181 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     6241 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     6301 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     6361 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     6421 cgaaaagtgc cacctgacgt c
//
