LOCUS       sequence-386891-        4810 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 0 PhiC31-intein(DnaE-Npu) with GG1 and GG4 golden gate 
            linkers.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4810)
  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 4810)
  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-I39 PhiC31-Intein GG1-GG4
FEATURES             Location/Qualifiers
     source          1..4810
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1256..1273
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1411..1427)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1411..1427)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1424..1446)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1435..1451
                     /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)."
     promoter        complement(join(1459..1465,1466..1483,1484..1489))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1504..1525
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1642..1659)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1813..2401)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1893..1912)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2539..2913)
                     /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"
     regulatory      2910..2919
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(3119..3913)
                     /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(3119..3913)
                     /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(3230..3249)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3840..3859
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(4262..4564)
                     /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     complement(4364..4383)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     4687..4709
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4701..4718
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4702..4718
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4725..4744
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4725..4743
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggatcggagg tctcaaatgg acacgtatgc cggcgcttat gatagacaat caagagaaag
       61 agagaactct tctgccgcct ctccagctac acagagatct gccaacgagg ataaagctgc
      121 tgatttacaa agagaggtag agagagatgg gggtaggttt agatttgtgg gtcatttctc
      181 tgaggctcct ggtacatcag cctttggtac tgctgaacgg cctgaatttg aaagaatatt
      241 aaatgagtgt cgggccggtc gactaaatat gataatagtt tatgatgtat ctaggttcag
      301 tagactcaaa gttatggatg caattccgat cgtgagcgag ctacttgcac tcggtgttac
      361 tatcgtaagt acacaagaag gtgtattcag gcagggtaat gtgatggacc ttatacacct
      421 tattatgcga ctcgacgcta gccataaaga gtcatcactg aagagtgcta aaatcttgga
      481 caccaagaac ctgcaaaggg agcttggagg ctatgtagga ggaaaagctc cgtatggttt
      541 cgaactcgtg agtgagacaa aagagattac cagaaatggt aggatggtga acgtggttat
      601 taataagcta gctcatagca caacccctct cacagggcct tttgagttcg aacctgacgt
      661 tattagatgg tggtggagag agatcaaaac tcacaaacat cttccattta agccaggtag
      721 ccaggctgct atccaccctg gatctatcac tgggctttgt aaaaggatgg atgctgacgc
      781 agtgccaact cgaggggaaa caatcggtaa gaagactgca tcttccgctt gggatcccgc
      841 tacagttatg agaatccttc gggatccgcg tattgctgga ttcgccgcag aagtcattta
      901 taagaaaaaa cctgatggta ctcctactac aaaaatcgaa tattgcctga gctatgaaac
      961 cgaaattctg accgtggaat atggcagcct gccgattggc aaaattgtgg aaaaacgcat
     1021 tgaatgcacc gtgtatagcg tggataacaa cggcaacatt tatacccagc cggtggcgca
     1081 gtggcatgat cgcggcgaac aggaagtgtt tgaatattgc ctggaagatg gcagcctgat
     1141 tcgcgcgacc aaagatcata aatttatgac cgtggatggc cagatgctgc cgattgatga
     1201 aatttttgaa cgcgaactgg atctgatgcg cgtggataac ctgccgaaca ccagccatca
     1261 tcatcatcat cattaataag ctttgagacc tcatcacgcc tgaattccag cacactggcg
     1321 gccgttacta gtggatccga gctcggtacc aagcttgatg catagcttga gtattctaac
     1381 gcgtcaccta aatagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     1441 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     1501 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     1561 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     1621 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     1681 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     1741 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     1801 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     1861 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     1921 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     1981 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     2041 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     2101 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     2161 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     2221 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     2281 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     2341 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     2401 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     2461 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     2521 atgaagtttt agcacgtgtc agtcctgctc ctcggccacg aagtgcacgc agttgccggc
     2581 cgggtcgcgc agggcgaact cccgccccca cggctgctcg ccgatctcgg tcatggccgg
     2641 cccggaggcg tcccggaagt tcgtggacac gacctccgac cactcggcgt acagctcgtc
     2701 caggccgcgc acccacaccc aggccagggt gttgtccggc accacctggt cctggaccgc
     2761 gctgatgaac agggtcacgt cgtcccggac cacaccggcg aagtcgtcct ccacgaagtc
     2821 ccgggagaac ccgagccggt cggtccagaa ctcgaccgct ccggcgacgt cgcgcgcggt
     2881 gagcaccgga acggcactgg tcaacttggc catggtggcc ctcctcacgt gctattattg
     2941 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     3001 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgatg cggtgtgaaa
     3061 taccgcacag atgcgtaagg agaaaatacc gcatcaggaa attgtaagcg ttaataattc
     3121 agaagaactc gtcaagaagg cgatagaagg cgatgcgctg cgaatcggga gcggcgatac
     3181 cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag ctcttcagca atatcacggg
     3241 tagccaacgc tatgtcctga tagcggtccg ccacacccag ccggccacag tcgatgaatc
     3301 cagaaaagcg gccattttcc accatgatat tcggcaagca ggcatcgcca tgggtcacga
     3361 cgagatcctc gccgtcgggc atgctcgcct tgagcctggc gaacagttcg gctggcgcga
     3421 gcccctgatg ctcttcgtcc agatcatcct gatcgacaag accggcttcc atccgagtac
     3481 gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg gcaggtagcc ggatcaagcg
     3541 tatgcagccg ccgcattgca tcagccatga tggatacttt ctcggcagga gcaaggtgag
     3601 atgacaggag atcctgcccc ggcacttcgc ccaatagcag ccagtccctt cccgcttcag
     3661 tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt ggccagccac gatagccgcg
     3721 ctgcctcgtc ttgcagttca ttcagggcac cggacaggtc ggtcttgaca aaaagaaccg
     3781 ggcgcccctg cgctgacagc cggaacacgg cggcatcaga gcagccgatt gtctgttgtg
     3841 cccagtcata gccgaatagc ctctccaccc aagcggccgg agaacctgcg tgcaatccat
     3901 cttgttcaat catgcgaaac gatcctcatc ctgtctcttg atcagagctt gatcccctgc
     3961 gccatcagat ccttggcggc aagaaagcca tccagtttac tttgcagggc ttcccaacct
     4021 taccagaggg cgccccagct ggcaattccg gttcgcttgc tgtccataaa accgcccagt
     4081 ctagctatcg ccatgtaagc ccactgcaag ctacctgctt tctctttgcg cttgcgtttt
     4141 cccttgtcca gatagcccag tagctgacat tcatccgggg tcagcaccgt ttctgcggac
     4201 tggctttcta cgtgaaaagg atctaggtga agatcctttt tgataatctc atgcctgaca
     4261 tttatattcc ccagaacatc aggttaatgg cgtttttgat gtcattttcg cggtggctga
     4321 gatcagccac ttcttccccg ataacggaga ccggcacact ggccatatcg gtggtcatca
     4381 tgcgccagct ttcatccccg atatgcacca ccgggtaaag ttcacgggag actttatctg
     4441 acagcagacg tgcactggcc agggggatca ccatccgtcg ccccggcgtg tcaataatat
     4501 cactctgtac atccacaaac agacgataac ggctctctct tttataggtg taaaccttaa
     4561 actgccgtac gtataggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
     4621 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
     4681 ggttttccca gtcacgacgt tgtaaaacga cggccagtga attgtaatac gactcactat
     4741 agggcgaatt gggccctcta gatgcatgct cgagcggccg ccagtgtgat ggatatctgc
     4801 agaattcagg
//
