LOCUS       sequence-214498-        7887 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains two recombination homology arms and loxP-flanked cassette 
            encoding puromycin resistance gene, Venus fluorescence marker, 
            thymidine kinase suicide gene, for scarless chromosomal engineering.
            .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7887)
  TITLE     Engineering scarless chromosomal rearrangements using 
            homology-mediated recombination and selection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7887)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pJ151-HDR
FEATURES             Location/Qualifiers
     source          1..7887
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     31..50
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    complement(761..794)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        811..1992
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1041..1983
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1940..1960
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        1994..2041
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     primer_bind     complement(2072..2091)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2568..2588
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             2669..2722
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(2729..2731,2732..2734,2735..3448)
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2774..2795)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3035..3054)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    join(3470..4043,4044..4046,4047..4055)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3637..3654)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3864..3883
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4056..5051
                     /codon_start=1
                     /product="truncated herpes simplex virus thymidine kinase 
                     (Salomon et al., 1995)"
                     /label=Delta-TK
                     /note="confers sensitivity to nucleoside analogs such as 
                     acyclovir or ganciclovir"
                     /translation="MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWRV
                     LGASETIANIYTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGS
                     SHAPPPALTLIFDRHPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGAL
                     PEDRHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQGGGSWREDWGQLSGTAV
                     PPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFI
                     LDYDQSPAGCRDALLQLTSGMIQTHVTTPGSIPTICDLARTFAREMGEAN"
     polyA_signal    5072..5193
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5109..5128)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5163..5182
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(5209..5242)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(5954..5971)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5954..5970)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6069..6086)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6240..6828)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6320..6339)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6950..7759)
                     /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"
     promoter        complement(7760..7851)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 acattatcgc gagcccattt atacctgaat atggctcata acaccccttg cagtgcgact
       61 aacggcatga agctcgtcgg ggctggatcg cttcgtattg cgagctgtgc ggctgagttg
      121 acgtatctgt gctggatgat tactcataac ggcaccgcta tcaaacgtgc cacgttcatg
      181 tccgtgtcgc ccttattcga ctcggggggc gcgcccatat gttcgaagga tccatttaaa
      241 taaatcacag catacctcac acagttttct tattgtaagg cttactcaat gatgcatgtt
      301 tttaatgtag tgatgatcat taaggttctt gctttattta ttataatagg tgatgaccca
      361 gttttcctct tgttagatat tgctgtgtgt ctagggacct gagactgttt tggtgtgatt
      421 ggcacttggt gctgtgctct gtcttgattc actttgtcac aatggtctct ctcctgtgag
      481 ctcctatgac cttgacagtt cttactcaga ctgctgttgc ctttgctgac ttcccagatg
      541 taccacaaac aaacctttaa ttctctcttc ctctacattc atacccttct aaacttatta
      601 ataactgaga atgtatttaa tgcattgaat tctactttaa aatgatttaa agtttatctt
      661 ctgtgtcttg ccacaattag gaaaagtaat gctactgttg aatttatttc ctctaaatat
      721 aacttgtcac attgaccatt tttaattaac gcgtgctagc ataacttcgt atagcataca
      781 ttatacgaag ttatcctagg taattctacc gctccggtgc ccgtcagtgg gcagagcgca
      841 catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga
      901 gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg
      961 agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg
     1021 ggtttgccgc cagaacacag gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta
     1081 cgggttatgg cccttgcgtg ccttgaatta cttccacgcc cctggctgca gtacgtgatt
     1141 cttgatcccg agcttcgggt tggaagtggg tgggagagtt cgaggccttg cgcttaagga
     1201 gccccttcgc ctcgtgcttg agttgaggcc tggcctgggc gctggggccg ccgcgtgcga
     1261 atctggtggc accttcgcgc ctgtctcgct gctttcgata agtctctagc catttaaaat
     1321 ttttgatgac ctgctgcgac gctttttttc tggcaagata gtcttgtaaa tgcgggccaa
     1381 gatctgcaca ctggtatttc ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc
     1441 cagcgcacat gttcggcgag gcggggcctg cgagcgcggc caccgagaat cggacggggg
     1501 tagtctcaag ctggccggcc tgctctggtg cctggcctcg cgccgccgtg tatcgccccg
     1561 ccctgggcgg caaggctggc ccggtcggca ccagttgcgt gagcggaaag atggccgctt
     1621 cccggccctg ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga gcgggcgggt
     1681 gagtcaccca cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc
     1741 acggagtacc gggcgccgtc caggcacctc gattagttct cgagcttttg gagtacgtcg
     1801 tctttaggtt ggggggaggg gttttatgcg atggagtttc cccacactga gtgggtggag
     1861 actgaagtta ggccagcttg gcacttgatg taattctcct tggaatttgc cctttttgag
     1921 tttggatctt ggttcattct caagcctcag acagtggttc aaagtttttt tcttccattt
     1981 caggtgtcgt gatgttgaca attaatcatc ggcatagtat atcggcatag tataatacga
     2041 caaggtgagg aactaaacca gatctgccac catgaccgag tacaagccca cggtgcgcct
     2101 cgccacccgc gacgacgtcc ctcgggctgt acgcaccctc gccgccgcgt tcgccgacta
     2161 ccccgccacg cgccacaccg tggacccgga ccgccacatc gagcgggtca ccgagctgca
     2221 agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg
     2281 cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga
     2341 gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga
     2401 aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt
     2461 ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc
     2521 ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt
     2581 ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac
     2641 ctggtgcatg acccgcaagg gaagcggaga gggcagagga agtcttctaa catgcggtga
     2701 cgtggaggag aatcccggcc ctggtaccat ggtgagcaag ggcgaggagc tgttcaccgg
     2761 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
     2821 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagctga tctgcaccac
     2881 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgggctacg gcctgcagtg
     2941 cttcgcccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     3001 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     3061 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     3121 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     3181 ctatatcacc gccgacaagc agaagaacgg catcaaggcc aacttcaaga tccgccacaa
     3241 catcgaggac ggcggcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     3301 cggccccgtg ctgctgcccg acaaccacta cctgagctac cagtccgccc tgagcaaaga
     3361 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     3421 tctcggcatg gacgagctgt acaagtaagc ggccgccccg gggcatgcgc ccctctccct
     3481 cccccccccc taacgttact ggccgaagcc gcttggaata aggccggtgt gcgtttgtct
     3541 atatgttatt ttccaccata ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc
     3601 ctgtcttctt gacgagcatt cctaggggtc tttcccctct cgccaaagga atgcaaggtc
     3661 tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa acaacgtctg
     3721 tagcgaccct ttgcaggcag cggaaccccc cacctggcga caggtgcctc tgcggccaaa
     3781 agccacgtgt ataagataca cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt
     3841 ggatagttgt ggaaagagtc aaatggctct cctcaagcgt attcaacaag gggctgaagg
     3901 atgcccagaa ggtaccccat tgtatgggat ctgatctggg gcctcggtgc acatgcttta
     3961 catgtgttta gtcgaggtta aaaaacgtct aggccccccg aaccacgggg acgtggtttt
     4021 cctttgaaaa acacgatgat aatatggcca caaccatgcc cacgctactg cgggtttata
     4081 tagacggtcc ccacgggatg gggaaaacca ccaccacgca actgctggtg gccctgggtt
     4141 cgcgcgacga tatcgtctac gtacccgagc cgatgactta ctggcgggtg ctgggggctt
     4201 ccgagacaat cgcgaacatc tacaccacac aacaccgcct tgaccagggt gagatatcgg
     4261 ccggggacgc ggcggtggta atgacaagcg cccagataac aatgggaatg ccttatgccg
     4321 tgaccgacgc cgttctggct cctcatatcg ggggggaggc tgggagctca catgccccgc
     4381 ccccggccct caccctcatc ttcgaccgcc atcccatcgc cgccctcctg tgctacccgg
     4441 ccgcgcgata ccttatgggc agcatgaccc cccaggccgt gctggcgttc gtggccctca
     4501 tcccgccgac cttgcccggc acaaacatcg tgttgggggc ccttccggag gacagacaca
     4561 tcgaccgcct ggccaaacgc cagcgccccg gcgagcggct tgacctggct atgctggccg
     4621 cgattcgccg cgtttacggg ctgcttgcca atacggtgcg gtatctgcag ggcggcgggt
     4681 cgtggcggga ggattgggga cagctttcgg ggacggccgt gccgccccag ggtgccgagc
     4741 cccagagcaa cgcgggccca cgaccccata tcggggacac gttatttacc ctgtttcggg
     4801 cccccgagtt gctggccccc aacggcgacc tgtacaacgt gtttgcctgg gccttggacg
     4861 tcttggccaa acgcctccgt cccatgcacg tctttatcct ggattacgac caatcgcccg
     4921 ccggctgccg ggacgccctg ctgcaactta cctccgggat gatccagacc cacgtcacca
     4981 ccccaggctc cataccgacg atctgcgacc tggcgcgcac gtttgcgcgg gagatggggg
     5041 aggctaactg acccggggca tgcgcggccg caacttgttt attgcagctt ataatggtta
     5101 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     5161 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgcaattgat aacttcgtat
     5221 agcatacatt atacgaagtt atatcgattg ttaactctag atatgggcta aaagaatact
     5281 ggcgccaata ttaagccaaa atctgtaata tgtctacaat tcttggttga ggtagagagt
     5341 ggctgtttgc aattaaatag tacccagagg cataacactt tctggctaga ttttggttaa
     5401 ggcaaacttt tgtgatatgt aaccaaatcc acttcactat taaaggctgt acttgtgata
     5461 tgagaagtga tcagctgtgg tagacagttg gttctgtact actgcatcca gacttgtgtt
     5521 ggttgtatat gtattatagt acttagtttt cattttaact tactttctaa ggaacactgg
     5581 aactgcccaa tttaatttca aaagttggag gattttgttt tgagacaaga tttccctctg
     5641 taagcctgtt ctgtcctcag ctccggagtg ctagggctct aggcgtgagc catcatgctg
     5701 aatttagatt agcatttaaa cagaaattat taaagcatag gaaagaagtc tcagtggtta
     5761 catttaaatg tcgacctgca ggcgtacgac tagtaaaaga gtcgaataag ggcgacacac
     5821 tgtcattagc aactccttgt ccttcgatct cgtcaacaac agcttgcagt tcaaatacaa
     5881 gacccagaag gcgactattc tggaagcgag cttgaagcca gacgctgagt acgaaaaggg
     5941 gcccgagctt aagactggcc gtcgttttac aacacagaaa gagtttgtag aaacgcaaaa
     6001 aggccatccg tcaggggcct tctgcttagt ttgatgcctg gcagttccct actctcgcct
     6061 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     6121 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     6181 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     6241 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     6301 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     6361 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     6421 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     6481 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     6541 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     6601 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtgggct
     6661 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     6721 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     6781 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     6841 atcttttcta cggggtctga cgctcagtgg aacgacgcgc gcgtaactca cgttaaggga
     6901 ttttggtcat gagcttgcgc cgtcccgtca agtcagcgta atgctctgct tagaaaaact
     6961 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
     7021 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
     7081 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
     7141 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
     7201 agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct
     7261 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
     7321 ggcgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgag tgcaaccggc
     7381 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata
     7441 cctggaacgc tgtttttccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
     7501 ggataaaatg cttgatggtc ggaagtggca taaattccgt cagccagttt agtctgacca
     7561 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
     7621 catcgggctt cccatacaag cgatagattg tcgcacctga ttgcccgaca ttatcgcgag
     7681 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctcgacgttt
     7741 cccgttgaat atggctcata ttcttccttt ttcaatatta ttgaagcatt tatcagggtt
     7801 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggtca
     7861 gtgttacaac caattaacca attctga
//
