LOCUS       sequence-351784-        4308 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cargo EGFP with AttP Bxb1 site for in-frame fusion (Parent 
            minicircle).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4308)
  AUTHORS   Yarnall MTN, Ioannidi EI, Schmitt-Ulms C, Krajeski RN, Lim J, 
            Villiger L, Zhou W, Jiang K, Garushyants SK, Roberts N, Zhang L, 
            Vakulskas CA, Walker JA 2nd, Kadina AP, Zepeda AE, Holden K, Ma H, 
            Xie J, Gao G, Foquet L, Bial G, Donnelly SK, Miyata Y, Radiloff DR, 
            Henderson JM, Ujita A, Abudayyeh OO, Gootenberg JS
  TITLE     Drag-and-drop genome insertion of large sequences without 
            double-strand DNA cleavage using CRISPR-directed integrases.
  JOURNAL   Nat Biotechnol. 2022 Nov 24. doi: 10.1038/s41587-022-01527-4.
  PUBMED    36424489
REFERENCE   2  (bases 1 to 4308)
  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 
            Biotechnol. 2022 Nov 24. doi: 10.1038/s41587-022-01527-4."
COMMENT     Sequence Label: pDY0423 Cargo EGFP with AttP Bxb1 site for in-...
FEATURES             Location/Qualifiers
     source          1..4308
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(328..345)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      498..584
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      676..703
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      837..1425
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1326..1345
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1579..1596
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1611..1751
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1677..1696)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1837..1859
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2538..2555)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    2576..2609
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     CDS             2619..3284
                     /codon_start=1
                     /product="green fluorescent protein 2 from Pontellina 
                     plumata, also known as ppluGFP2 (Shagin et al., 2004)"
                     /label=CopGFP
                     /translation="MPAMKIECRITGTLNGVEFELVGGGEGTPEQGRMTNKMKSTKGAL
                     TFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYR
                     YEAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDG
                     GYYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEYQHAFKTPIAFA
                     "
     CDS             2619..3284
                     /codon_start=1
                     /product="green fluorescent protein 2 from Pontellina 
                     plumata, also known as ppluGFP2 (Shagin et al., 2004)"
                     /label=CopGFP
                     /translation="MPAMKIECRITGTLNGVEFELVGGGEGTPEQGRMTNKMKSTKGAL
                     TFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYR
                     YEAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDG
                     GYYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEYQHAFKTPIAFA
                     "
     protein_bind    3303..3350
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attP site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             complement(3478..4272)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(3478..4272)
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3589..3608)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4199..4218
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 tgatcccctg cgccatcaga tccttggcgg caagaaagcc atccagttta ctttgcaggg
       61 cttcccaacc ttaccagagg gcgccccagc tggcaattcc ggttcgcttg ctgtccataa
      121 aaccgcccag tctagctatc gccatgtaag cccactgcaa gctacctgct ttctctttgc
      181 gcttgcgttt tcccttgtcc agatagccca gtagctgaca ttcatccggg gtcagcaccg
      241 tttctgcgga ctggctttct acgcgctcga gggggggcca aacggtctcc agcttggctg
      301 ttttggcgga tgagagaaga ttttcagcct gatacagatt aaatcagaac gcagaagcgg
      361 tctgataaaa cagaatttgc ctggcggcag tagcgcggtg gtcccacctg accccatgcc
      421 gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg gggtctcccc atgcgagagt
      481 agggaactgc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt
      541 ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ggagcggatt
      601 tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg acgcccgcca taaactgcca
      661 ggcatcaaat taagcagaag gccatcctga cggatggcct ttttgcgttt ctacaaactc
      721 ttttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaccaa aatcccttaa
      781 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
      841 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
      901 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
      961 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     1021 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     1081 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     1141 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     1201 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     1261 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     1321 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     1381 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     1441 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     1501 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     1561 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     1621 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca
     1681 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg
     1741 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     1801 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     1861 tttcaccgtc atcaccgaaa cgcgcgaggc agcagatcaa ttcgcgcgcg aaggcgaagc
     1921 ggcatgcata atgtgcctgt caaatggacg aagcagggat tctgcaaacc ctatgctact
     1981 ccgtcaagcc gtcaattgtc tgattcgtta ccaattatga caacttgacg gctacatcat
     2041 tcactttttc ttcacaaccg gcacggaact cgctcgggct ggccccggtg cattttttaa
     2101 atacccgcga gaaatagagt tgatcgtcaa aaccaacatt gcgaccgacg gtggcgatag
     2161 gcatccgggt ggtgctcaaa agcagcttcg cctggctgat acgttggtcc tcgcgccagc
     2221 ttaagacgct aatccctaac tgctggcgga aaagatgtga cagacgcgac ggcgacaagc
     2281 aaacatgctg tgcgacgctg gcgattcttc tctcatccgc caaaacagcc aagctggaga
     2341 ccgtttgaca ttaccctgtt atccctagat acattaccct gttatcccag atgacatacc
     2401 ctgttatccc tagatgacat taccctgtta tcccagatga cattaccctg ttatccctag
     2461 atacattacc ctgttatccc agatgacata ccctgttatc cctagatgac attaccctgt
     2521 tatcccagat aaactcaatg atgatgatga tgatggtcga gactcagcgg ccgcggtgcc
     2581 agggcgtgcc cttgggctcc ccgggcgcgg ccgccaccat gcccgccatg aagatcgagt
     2641 gccgcatcac cggcaccctg aacggcgtgg agttcgagct ggtgggcggc ggagagggca
     2701 cccccgagca gggccgcatg accaacaaga tgaagagcac caaaggcgcc ctgaccttca
     2761 gcccctacct gctgagccac gtgatgggct acggcttcta ccacttcggc acctacccca
     2821 gcggctacga gaaccccttc ctgcacgcca tcaacaacgg cggctacacc aacacccgca
     2881 tcgagaagta cgaggacggc ggcgtgctgc acgtgagctt cagctaccgc tacgaggccg
     2941 gccgcgtgat cggcgacttc aaggtggtgg gcaccggctt ccccgaggac agcgtgatct
     3001 tcaccgacaa gatcatccgc agcaacgcca ccgtggagca cctgcacccc atgggcgata
     3061 acgtgctggt gggcagcttc gcccgcacct tcagcctgcg cgacggcggc tactacagct
     3121 tcgtggtgga cagccacatg cacttcaaga gcgccatcca ccccagcatc ctgcagaacg
     3181 ggggccccat gttcgccttc cgccgcgtgg aggagctgca cagcaacacc gagctgggca
     3241 tcgtggagta ccagcacgcc ttcaagaccc ccatcgcctt cgccagatct cgagctcgag
     3301 gtggtttgtc tggtcaacca ccgcggtctc agtggtgtac ggtacaaacc caccccaact
     3361 ggggtaacct ttgagttctc tcagttgggg gtaatcagca tcatgatgtg gtaccacatc
     3421 atgatgctga ttataagaat gcggccgcca cactctagtg gatctcgagt taataattca
     3481 gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag cggcgatacc
     3541 gtaaagcacg aggaagcggt cagcccattc gccgccaagc tcttcagcaa tatcacgggt
     3601 agccaacgct atgtcctgat agcggtccgc cacacccagc cggccacagt cgatgaatcc
     3661 agaaaagcgg ccattttcca ccatgatatt cggcaagcag gcatcgccat gggtcacgac
     3721 gagatcctcg ccgtcgggca tgctcgcctt gagcctggcg aacagttcgg ctggcgcgag
     3781 cccctgatgc tcttcgtcca gatcatcctg atcgacaaga ccggcttcca tccgagtacg
     3841 tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt
     3901 atgcagccgc cgcattgcat cagccatgat ggatactttc tcggcaggag caaggtgaga
     3961 tgacaggaga tcctgccccg gcacttcgcc caatagcagc cagtcccttc ccgcttcagt
     4021 gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc
     4081 tgcctcgtct tgcagttcat tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg
     4141 gcgcccctgc gctgacagcc ggaacacggc ggcatcagag cagccgattg tctgttgtgc
     4201 ccagtcatag ccgaatagcc tctccaccca agcggccgga gaacctgcgt gcaatccatc
     4261 ttgttcaatc atgcgaaacg atcctcatcc tgtctcttga tcagagct
//
