LOCUS       sequence-476062-        4490 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mini donor expressing GFP, used for PCR to generate IVT template..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4490)
  AUTHORS   Edmonds KK, Wilkinson ME, Strebinger D, Chen H, Lash B, Schaefer CC,
            Zhu S, Liu D, Zilberzwige-Tal S, Ladha A, Walsh ML, Frangieh CJ, Vaz
            Reay NA, Macrae RK, Wang X, Zhang F
  TITLE     Structure and biochemistry-guided engineering of an all-RNA system 
            for DNA insertion with R2 retrotransposons.
  JOURNAL   Nat Commun. 2025 Jul 2;16(1):6079. doi: 10.1038/s41467-025-61321-z.
  PUBMED    40603868
REFERENCE   2  (bases 1 to 4490)
  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: "Nat 
            Commun."; date: "2025-07-2"; volume: "16(1):6079. doi"; pages: " 
            10.1038/s41467-025-61321-z"
COMMENT     Sequence Label: pTmpl-miniDonor-GFP
FEATURES             Location/Qualifiers
     source          1..4490
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        27..131
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(132..200,201..992)
                     /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(350..369)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1163..1751
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1652..1671
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1905..1922
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2039..2060
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2075..2080,2081..2098,2099..2105)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2113..2129
                     /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     2118..2140
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2137..2153
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2137..2153
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        2276..2655
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2656..2859
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2810..2830
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2888..2897
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2894..2896,2897..2899,2900..3613)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2894..2896,2897..2899,2900..3610)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2939..2960)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3200..3219)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3547..3568
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3614..3662
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(3878..3895)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3878..3894)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3887..3909)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(4035..4490)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4172..4193)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4384..4403
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
       61 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
      121 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
      181 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
      241 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
      301 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
      361 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
      421 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
      481 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
      541 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
      601 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
      661 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
      721 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
      781 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
      841 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
      901 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
      961 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     1021 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     1081 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     1141 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     1201 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     1261 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     1321 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     1381 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     1441 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     1501 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     1561 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     1621 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     1681 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     1741 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     1801 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     1861 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     1921 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     1981 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     2041 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
     2101 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgagg
     2161 cgcgccgttg acgcgatgtg atttctgccc agtgctctga atgtcaaagt gaagaaattc
     2221 aatgaagcgc gggtaaacgg cgggagtaac tatgactctc ttaaggggat ccgttgacat
     2281 tgattattga ctagttatta atagtaatca attacggggt cattagttca tagcccatat
     2341 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
     2401 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc
     2461 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
     2521 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
     2581 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
     2641 atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt
     2701 gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac
     2761 caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc
     2821 ggtaggcgtg tacggtggga ggtctatata agcagagctc tctggctaac tagagaaccc
     2881 actgcttgcc accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
     2941 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
     3001 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     3061 gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc
     3121 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     3181 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     3241 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     3301 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga
     3361 caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag
     3421 cgtgcagctg gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     3481 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg
     3541 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     3601 gctgtacaag taaaataaaa gatctttatt ttcattagat ctgtgtgttg gttttttgtg
     3661 tgaagcttca cctgatttgg accaattcac gggatttgtc caaggtggac gggccacctt
     3721 tacttaaccc ggaaaaggaa catatataat ttatgtgtgt tcgataaata gccaaatgcc
     3781 tcgtcatcta attagtgacg cgcatgaatg gatgaacgag attcccactg tccctaccta
     3841 ctatccagcg aaaccacagc caagggaagc ggccgccact ggccgtcgtt ttacaacgtc
     3901 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     3961 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     4021 tgaatggcga atggaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg
     4081 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     4141 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     4201 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     4261 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     4321 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     4381 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     4441 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
//
