LOCUS       sequence-395042-        5677 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 0 PhiC31 target: attP_PhiC31-pUBQ10-attB_PhiC31 with golden 
            gate linker in pBLUNT plasmid..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5677)
  AUTHORS   Guiziou S, Maranas CJ, Chu JC, Nemhauser JL
  TITLE     An integrase toolbox to record gene-expression during plant 
            development.
  JOURNAL   Nat Commun. 2023 Apr 3;14(1):1844. doi: 10.1038/s41467-023-37607-5.
  PUBMED    37012288
REFERENCE   2  (bases 1 to 5677)
  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: "2023-04-3"; volume: "14(1):1844. doi"; pages: " 
            10.1038/s41467-023-37607-5"
COMMENT     Sequence Label: L0-T1 Target PhiC31
FEATURES             Location/Qualifiers
     source          1..5677
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      116..704
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     605..624
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     858..875
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    992..1013
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1028..1033,1034..1051,1052..1058)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1066..1082
                     /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     1071..1093
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1090..1106
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1090..1106
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    3327..3360
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     primer_bind     complement(3450..3469)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3451..3469)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3476..3493)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3476..3492)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3485..3507)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             3630..3932
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     3811..3830
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             4281..5075
                     /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             4281..5075
                     /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(4335..4354)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4945..4964
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      5275..5284
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5281..5655
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
ORIGIN
        1 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
       61 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
      121 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
      181 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
      241 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
      301 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
      361 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
      421 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
      481 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
      541 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
      601 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
      661 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
      721 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
      781 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
      841 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
      901 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
      961 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
     1021 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
     1081 aatttcacac aggaaacagc tatgaccatg attacgccaa gctatttagg tgacgcgtta
     1141 gaatactcaa gctatgcatc aagcttggta ccgagctcgg atccactagt aacggccgcc
     1201 agtgtgctgg aattcaggca ctctgtggtc tcactatagt agtgccccaa ctggggtaac
     1261 ctttgagttc tctcagttgg gggcgtaggg ctcaacaaca aactttccat tcacccattt
     1321 atggatacac agtctagctc aacagagctt ttaacccaaa ttggtacaat agaatacaac
     1381 tttagatcat aattctcaaa agaaagagat tccttagcta ttctatctgc cactccattt
     1441 ccttctcggc ttgtatgcac aagcataaaa tcctcaaact tgctaagtag atactttatg
     1501 tcttggataa ttggattgag acttgacaag cataactttc atgtaaccaa agacacaagt
     1561 tgctgagaat ccacctcaaa aatgatcttc ctataattga atcgggataa tgacagcaca
     1621 gcccatctaa gagcctccac ttctacttcc agcacgcttc ttacttttac cacagctctt
     1681 gcacctaacc ataacacctt ccctgtatga tcgcgaagca cccaccctaa gccacatttt
     1741 aatccttctg ttggccatgc cccatcaaag ttgcacttaa cccaagattg tggtggagct
     1801 tcccatgttt ctcgtctgtc ccgacggtgt tgtggttggt gctttcctta cattctgagc
     1861 ctctttcctt ctaatccact catctgcatc ttcttgtgtc cttactaata cctcattggt
     1921 tccaaattcc ctccctttaa gcaccagctc gtttctgttc ttccacagcc tcccaagtat
     1981 ccaagggact aaagcctcca cattcttcag atcaggatat tcttgtttaa gatgttgaac
     2041 tctatggagg tttgtatgaa ctgatgatct aggaccggat aagttccctt cttcatagcg
     2101 aacttattca aagaatgttt tgtgtatcat tcttgttaca ttgttattaa tgaaaaaata
     2161 ttattggtca ttggactgaa cacgagtgtt aaatatggac caggccccaa ataagatcca
     2221 ttgatatatg aattaaataa caagaataaa tcgagtcacc aaaccacttg ccttttttaa
     2281 cgagacttgt tcaccaactt gatacaaaag tcattatcct atgcaaatca ataatcatac
     2341 aaaaatatcc aataacacta aaaaattaaa agaaatggat aatttcacaa tatgttatac
     2401 gataaagaag ttacttttcc aagaaattca ctgattttat aagcccactt gcattagata
     2461 aatggcaaaa aaaaacaaaa aggaaaagaa ataaagcacg aagaattcta gaaaatacga
     2521 aatacgcttc aatgcagtgg gacccacggt tcaattattg ccaattttca gctccaccgt
     2581 atatttaaaa aataaaacga taatgctaaa aaaatataaa tcgtaacgat cgttaaatct
     2641 caacggctgg atcttatgac gaccgttaga aattgtggtt gtcgacgagt cagtaataaa
     2701 cggcgtcaaa gtggttgcag ccggcacaca cgagtcgtgt ttatcaactc aaagcacaaa
     2761 tacttttcct caacctaaaa ataaggcaat tagccaaaaa caactttgcg tgtaaacaac
     2821 gctcaataca cgtgtcattt tattattagc tattgcttca ccgccttagc tttctcgtga
     2881 cctagtcgtc ctcgtctttt cttcttcttc ttctataaaa caatacccaa agagctcttc
     2941 ttcttcacaa ttcagatttc aatttctcaa aatcttaaaa actttctctc aattctctct
     3001 accgtgatca aggtaaattt ctgtgttcct tattctctca aaatcttcga ttttgttttc
     3061 gttcgatccc aatttcgtat atgttctttg gtttagattc tgttaatctt agatcgaaga
     3121 cgattttctg ggtttgatcg ttagatatca tcttaattct cgattagggt ttcatagata
     3181 tcatccgatt tgttcaaata atttgagttt tgtcgaataa ttactcttcg atttgtgatt
     3241 tctatctaga tctggtgtta gtttctagtt tgtgcgatcg aatttgtcga ttaatctgag
     3301 tttttctgat taacagaggt ggagtacgcg cccggggagc ccaagggcac gccctggcac
     3361 ccgcaccgcg gaccttgaga cctcctgaat tctgcagata tccatcacac tggcggccgc
     3421 tcgagcatgc atctagaggg cccaattcgc cctatagtga gtcgtattac aattcactgg
     3481 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     3541 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     3601 cccaacagtt gcgcagccta tacgtacggc agtttaaggt ttacacctat aaaagagaga
     3661 gccgttatcg tctgtttgtg gatgtacaga gtgatattat tgacacgccg gggcgacgga
     3721 tggtgatccc cctggccagt gcacgtctgc tgtcagataa agtctcccgt gaactttacc
     3781 cggtggtgca tatcggggat gaaagctggc gcatgatgac caccgatatg gccagtgtgc
     3841 cggtctccgt tatcggggaa gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa
     3901 acgccattaa cctgatgttc tggggaatat aaatgtcagg catgagatta tcaaaaagga
     3961 tcttcaccta gatccttttc acgtagaaag ccagtccgca gaaacggtgc tgaccccgga
     4021 tgaatgtcag ctactgggct atctggacaa gggaaaacgc aagcgcaaag agaaagcagg
     4081 tagcttgcag tgggcttaca tggcgatagc tagactgggc ggttttatgg acagcaagcg
     4141 aaccggaatt gccagctggg gcgccctctg gtaaggttgg gaagccctgc aaagtaaact
     4201 ggatggcttt cttgccgcca aggatctgat ggcgcagggg atcaagctct gatcaagaga
     4261 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg
     4321 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
     4381 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
     4441 ccggtgccct gaatgaactg caagacgagg cagcgcggct atcgtggctg gccacgacgg
     4501 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
     4561 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
     4621 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
     4681 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
     4741 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
     4801 tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc
     4861 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
     4921 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg
     4981 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
     5041 tcgccttcta tcgccttctt gacgagttct tctgaattat taacgcttac aatttcctga
     5101 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatcagg tggcactttt
     5161 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     5221 ccgctcatga gacaataacc ctgataaatg cttcaataat agcacgtgag gagggccacc
     5281 atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc
     5341 gagttctgga ccgaccggct cgggttctcc cgggacttcg tggaggacga cttcgccggt
     5401 gtggtccggg acgacgtgac cctgttcatc agcgcggtcc aggaccaggt ggtgccggac
     5461 aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag
     5521 gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat cggcgagcag
     5581 ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca cttcgtggcc
     5641 gaggagcagg actgacacgt gctaaaactt cattttt
//
