LOCUS       sequence-98430-e        4637 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PCR template for the insertion of eGFP by en passant (two-step 
            Red-mediated) mutagenesis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4637)
  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 4637)
  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: pEP-EGFPin
FEATURES             Location/Qualifiers
     source          1..4637
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     2..18
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2..18
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     32..51
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        32..50
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(112..133)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     461..480
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             complement(730..1545)
                     /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             complement(730..1545)
                     /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     1453..1472
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1537..1556
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     1790..1811
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1888..1905)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1888..1904)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1897..1919)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2046..2502)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2184..2205)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2396..2415
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2529..2633
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2634..2702,2703..3494)
                     /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     complement(2852..2871)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3665..4253
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4154..4173
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4407..4424
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4541..4562
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4577..4582,4583..4600,4601..4607)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4615..4631
                     /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)."
     primer_bind     join(4620..4637,1..5)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 acaggaaaca gctatgacat gattacgaat ttaatacgac tcactatagg gaattcgagc
       61 tcggtacccc ttgtacagct cgtccatgcc gagagtgatc ccggcggcgg tcacgaactc
      121 cagcaggacc atgtgatcgc gcttctcgtt ggggtctttg ctcagggcgg actgggtgct
      181 caggtagtgg ttgtcgggca gcagcacggg gccgtcgccg atgggggtgt tctgctggta
      241 gtggtcggcg agctgcacgc tgccgtcctc gatgttgtgg cggatcttga agttcacctt
      301 gatgccgttc ttctgcttgt cggccatgat atagacgttg tggctgttgt agttgtactc
      361 cagcttgtgc cccaggatgt tgccgtcctc cttgaagtcg atgcccttca gctcgatgcg
      421 gttcaccagg gtgtcgccct cgaacttcac ctcggcgcgg gtcttgtagt tgccgtcgtc
      481 cttgaagaag atggtgcgct cctggacgta gccttcgggc atggcggact tgaagaagtc
      541 gtgctgcttc atgtggtcgg ggtagcggct gaagcactgc acgccgtagg tcagggtggt
      601 cacgagggtg ggccagggca cgggcagctt gccggtggtg cagatgaact tcagggtcag
      661 cttgccgtag gtggcatcgc cctcgccctc gccgactagc cagtgttaca accaattaac
      721 caattctgat tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg
      781 attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag
      841 gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc
      901 aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
      961 agtgacgact gaatccggtg agaatggcaa aagcttatgc atttctttcc agacttgttc
     1021 aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat
     1081 tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac
     1141 aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga
     1201 atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa
     1261 ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
     1321 cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg
     1381 tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga
     1441 ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
     1501 taatcgcggc ctcgagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt
     1561 actgtttatg taagcagaca gttttattgt tcatgatgat atatttttat cttgtgcaat
     1621 gtaacatcag agattttgag acacaacgtg gctttgttga ataaatcgat taccctgtta
     1681 tccctagcta gtggcagctt gccggtggtg cagatgaact tcagggtcag cttgccgtag
     1741 gtggcatcgc cctcgccctc gccggacacg ctgaacttgt ggccgtttac gtcgccgtcc
     1801 agctcgacca ggatgggcac caccccggtg aacagctcct cgcccttgct caccatggat
     1861 cctcgactgc aggcatgcaa gcttggcact ggccgtcgtt ttacaacgtc gtgactggga
     1921 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     1981 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     2041 atgggaaatt gtaaacgttt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     2101 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     2161 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     2221 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     2281 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     2341 ggatgccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     2401 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     2461 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg
     2521 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     2581 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     2641 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     2701 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     2761 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     2821 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     2881 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     2941 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     3001 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     3061 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     3121 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     3181 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     3241 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     3301 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     3361 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     3421 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     3481 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     3541 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     3601 tcccttaacg tgagttttcg ttccacggcg cgccagaccc cgtagaaaag atcaaaggat
     3661 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     3721 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     3781 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     3841 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     3901 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     3961 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     4021 cgacctacac cgaactgaga tacctacagc gtgagcattg agaaagcgcc acgcttcccg
     4081 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     4141 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     4201 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     4261 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     4321 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     4381 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     4441 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     4501 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
     4561 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     4621 gcggataaca atttcac
//
