LOCUS       sequence-60186-e        4415 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4415)
  AUTHORS   Dolt KS, Lawrence ML, Miller-Hodges E, Slight J, Thornburn A, 
            Devenney PS, Hohenstein P
  TITLE     A universal vector for high-efficiency multi-fragment recombineering
            of BACs and knock-in constructs.
  JOURNAL   PLoS One. 2013 Apr 29;8(4):e62054. doi: 
            10.1371/journal.pone.0062054. Print 2013.
  PUBMED    23637962
REFERENCE   2  (bases 1 to 4415)
  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: "PLoS One.";
            date: "2013-04-29"; volume: "8(4):e62054. doi"; pages: " 
            10.1371/journal.pone.0062054. Print 2013"
COMMENT     Sequence Label: pENTR eGFPCRE L1-R5
FEATURES             Location/Qualifiers
     source          1..4415
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(171..190)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(172..190)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(195..211)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(195..211)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(313..332)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             324..1133
                     /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="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(391..410)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1280..1868
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1769..1788
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2022..2039
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2198..2225)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2317..2403)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2452..2474
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2466..2483
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2467..2483
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2499..2598
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             join(2604..2606,2607..2609,2610..3320)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2604..2606,2607..2609,2610..3320)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2910..2929)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3257..3278
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3375..4406
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="MANLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
                     LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
                     PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
                     LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
                     RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
                     DGD"
     protein_bind    join(4408..4415,1..116)
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 tatacaaaag ttgaacgaga aacgtaaaat gatataaata tcaatatatt aaattagatt
       61 ttgcataaaa aacagactac ataatactgt aaaacacaac atatccagtc actatgaatc
      121 aactacttag atggtattag tgacctgtac tgcagggcgg ccgcgatatc ccctatagtg
      181 agtcgtatta catggtcata gctgtttcct ggcagctctg gcccgtgtct caaaatctct
      241 gatgttacat tgcacaagat aaaaatatat catcatgaac aataaaactg tctgcttaca
      301 taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtcg aggccgcgat
      361 taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc
      421 aatcaggtgc gacaatctat cgcttgtatg ggaagcccga tgcgccagag ttgtttctga
      481 aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc
      541 tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat
      601 ggttactcac cactgcgatc cccggaaaaa cagcattcca ggtattagaa gaatatcctg
      661 attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc
      721 ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac
      781 gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg
      841 ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat tcagtcgtca
      901 ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta
      961 ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact
     1021 gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata
     1081 atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc taatcagaat
     1141 tggttaattg gttgtaacac tggcagagca ttacgctgac ttgacgggac ggcgcaagct
     1201 catgaccaaa atcccttaac gtgagttacg cgtcgttcca ctgagcgtca gaccccgtag
     1261 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     1321 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     1381 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     1441 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     1501 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     1561 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     1621 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     1681 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     1741 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     1801 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     1861 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     1921 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     1981 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     2041 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     2101 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata
     2161 cgcgtaccgc tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc
     2221 cttctgctta gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg
     2281 ccgttgcttc acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt
     2341 caccgacaaa caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat
     2401 ttgatgcctg gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac
     2461 gacgttgtaa aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac
     2521 tgatagtgac ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt
     2581 tgtacaaaaa agcaggctta accatggtga gcaagggcga ggagctgttc accggggtgg
     2641 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     2701 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     2761 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
     2821 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     2881 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     2941 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     3001 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     3061 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     3121 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     3181 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca
     3241 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     3301 gcatggacga gctgtacaag tccggtacag ctctcgacgg agaaagctca ggctctggct
     3361 cagagtctga ctccatggcc aatttactga ccgtacacca aaatttgcct gcattaccgg
     3421 tcgatgcaac gagtgatgag gttcgcaaga acctgatgga catgttcagg gatcgccagg
     3481 cgttttctga gcatacctgg aaaatgcttc tgtccgtttg ccggtcgtgg gcggcatggt
     3541 gcaagttgaa taaccggaaa tggtttcccg cagaacctga agatgttcgc gattatcttc
     3601 tatatcttca ggcgcgcggt ctggcagtaa aaactatcca gcaacatttg ggccagctaa
     3661 acatgcttca tcgtcggtcc gggctgccac gaccaagtga cagcaatgct gtttcactgg
     3721 ttatgcggcg gatccgaaaa gaaaacgttg atgccggtga acgtgcaaaa caggctctag
     3781 cgttcgaacg cactgatttc gaccaggttc gttcactcat ggaaaatagc gatcgctgcc
     3841 aggatatacg taatctggca tttctgggga ttgcttataa caccctgtta cgtatagccg
     3901 aaattgccag gatcagggtt aaagatatct cacgtactga cggtgggaga atgttaatcc
     3961 atattggcag aacgaaaacg ctggttagca ccgcaggtgt agagaaggca cttagcctgg
     4021 gggtaactaa actggtcgag cgatggattt ccgtctctgg tgtagctgat gatccgaata
     4081 actacctgtt ttgccgggtc agaaaaaatg gtgttgccgc gccatctgcc accagccagc
     4141 tatcaactcg cgccctggaa gggatttttg aagcaactca tcgattgatt tacggcgcta
     4201 aggatgactc tggtcagaga tacctggcct ggtctggaca cagtgcccgt gtcggagccg
     4261 cgcgagatat ggcccgcgct ggagtttcaa taccggagat catgcaagct ggtggctgga
     4321 ccaatgtaaa tattgtcatg aactatatcc gtaacctgga tagtgaaaca ggggcaatgg
     4381 tgcgcctgct ggaagatggc gattagacaa ctttg
//
