LOCUS       sequence-222216-        7885 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 7885)
  TITLE     Engineering scarless chromosomal rearrangements using 
            homology-mediated recombination and selection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7885)
  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            "Unpublished"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..7885
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     31..50
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    complement(759..792)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        809..1990
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1039..1981
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1938..1958
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        1992..2039
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     primer_bind     complement(2070..2089)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2566..2586
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             2667..2720
                     /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(2727..2729,2730..2732,2733..3446)
                     /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(2772..2793)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3033..3052)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    join(3468..4041,4042..4044,4045..4053)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3635..3652)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3862..3881
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4054..5049
                     /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    5070..5191
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5107..5126)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5161..5180
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(5207..5240)
                     /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(5952..5969)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5952..5968)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6067..6084)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6238..6826)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6318..6337)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6948..7757)
                     /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(7758..7849)
                     /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 ctcggggcgc gcccatatgt tcgaaggatc catttaaata
      241 aatcacagca tacctcacac agttttctta ttgtaaggct tactcaatga tgcatgtttt
      301 taatgtagtg atgatcatta aggttcttgc tttatttatt ataataggtg atgacccagt
      361 tttcctcttg ttagatattg ctgtgtgtct agggacctga gactgttttg gtgtgattgg
      421 cacttggtgc tgtgctctgt cttgattcac tttgtcacaa tggtctctct cctgtgagct
      481 cctatgacct tgacagttct tactcagact gctgttgcct ttgctgactt cccagatgta
      541 ccacaaacaa acctttaatt ctctcttcct ctacattcat acccttctaa acttattaat
      601 aactgagaat gtatttaatg cattgaattc tactttaaaa tgatttaaag tttatcttct
      661 gtgtcttgcc acaattagga aaagtaatgc tactgttgaa tttatttcct ctaaatataa
      721 cttgtcacat tgaccatttt taattaacgc gtgctagcat aacttcgtat agcatacatt
      781 atacgaagtt atcctaggta attctaccgc tccggtgccc gtcagtgggc agagcgcaca
      841 tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga
      901 aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag
      961 ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg
     1021 tttgccgcca gaacacaggt aagtgccgtg tgtggttccc gcgggcctgg cctctttacg
     1081 ggttatggcc cttgcgtgcc ttgaattact tccacgcccc tggctgcagt acgtgattct
     1141 tgatcccgag cttcgggttg gaagtgggtg ggagagttcg aggccttgcg cttaaggagc
     1201 cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc tggggccgcc gcgtgcgaat
     1261 ctggtggcac cttcgcgcct gtctcgctgc tttcgataag tctctagcca tttaaaattt
     1321 ttgatgacct gctgcgacgc tttttttctg gcaagatagt cttgtaaatg cgggccaaga
     1381 tctgcacact ggtatttcgg tttttggggc cgcgggcggc gacggggccc gtgcgtccca
     1441 gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca ccgagaatcg gacgggggta
     1501 gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg ccgccgtgta tcgccccgcc
     1561 ctgggcggca aggctggccc ggtcggcacc agttgcgtga gcggaaagat ggccgcttcc
     1621 cggccctgct gcagggagct caaaatggag gacgcggcgc tcgggagagc gggcgggtga
     1681 gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc gtcgcttcat gtgactccac
     1741 ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg agcttttgga gtacgtcgtc
     1801 tttaggttgg ggggaggggt tttatgcgat ggagtttccc cacactgagt gggtggagac
     1861 tgaagttagg ccagcttggc acttgatgta attctccttg gaatttgccc tttttgagtt
     1921 tggatcttgg ttcattctca agcctcagac agtggttcaa agtttttttc ttccatttca
     1981 ggtgtcgtga tgttgacaat taatcatcgg catagtatat cggcatagta taatacgaca
     2041 aggtgaggaa ctaaaccaga tctgccacca tgaccgagta caagcccacg gtgcgcctcg
     2101 ccacccgcga cgacgtccct cgggctgtac gcaccctcgc cgccgcgttc gccgactacc
     2161 ccgccacgcg ccacaccgtg gacccggacc gccacatcga gcgggtcacc gagctgcaag
     2221 aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg
     2281 ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga
     2341 tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag
     2401 gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct
     2461 cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg
     2521 ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct
     2581 acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct
     2641 ggtgcatgac ccgcaaggga agcggagagg gcagaggaag tcttctaaca tgcggtgacg
     2701 tggaggagaa tcccggccct ggtaccatgg tgagcaaggg cgaggagctg ttcaccgggg
     2761 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     2821 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagctgatc tgcaccaccg
     2881 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gggctacggc ctgcagtgct
     2941 tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     3001 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     3061 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     3121 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     3181 atatcaccgc cgacaagcag aagaacggca tcaaggccaa cttcaagatc cgccacaaca
     3241 tcgaggacgg cggcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     3301 gccccgtgct gctgcccgac aaccactacc tgagctacca gtccgccctg agcaaagacc
     3361 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     3421 tcggcatgga cgagctgtac aagtaagcgg ccgccccggg gcatgcgccc ctctccctcc
     3481 ccccccccta acgttactgg ccgaagccgc ttggaataag gccggtgtgc gtttgtctat
     3541 atgttatttt ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct
     3601 gtcttcttga cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg
     3661 ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta
     3721 gcgacccttt gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag
     3781 ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg
     3841 atagttgtgg aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat
     3901 gcccagaagg taccccattg tatgggatct gatctggggc ctcggtgcac atgctttaca
     3961 tgtgtttagt cgaggttaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc
     4021 tttgaaaaac acgatgataa tatggccaca accatgccca cgctactgcg ggtttatata
     4081 gacggtcccc acgggatggg gaaaaccacc accacgcaac tgctggtggc cctgggttcg
     4141 cgcgacgata tcgtctacgt acccgagccg atgacttact ggcgggtgct gggggcttcc
     4201 gagacaatcg cgaacatcta caccacacaa caccgccttg accagggtga gatatcggcc
     4261 ggggacgcgg cggtggtaat gacaagcgcc cagataacaa tgggaatgcc ttatgccgtg
     4321 accgacgccg ttctggctcc tcatatcggg ggggaggctg ggagctcaca tgccccgccc
     4381 ccggccctca ccctcatctt cgaccgccat cccatcgccg ccctcctgtg ctacccggcc
     4441 gcgcgatacc ttatgggcag catgaccccc caggccgtgc tggcgttcgt ggccctcatc
     4501 ccgccgacct tgcccggcac aaacatcgtg ttgggggccc ttccggagga cagacacatc
     4561 gaccgcctgg ccaaacgcca gcgccccggc gagcggcttg acctggctat gctggccgcg
     4621 attcgccgcg tttacgggct gcttgccaat acggtgcggt atctgcaggg cggcgggtcg
     4681 tggcgggagg attggggaca gctttcgggg acggccgtgc cgccccaggg tgccgagccc
     4741 cagagcaacg cgggcccacg accccatatc ggggacacgt tatttaccct gtttcgggcc
     4801 cccgagttgc tggcccccaa cggcgacctg tacaacgtgt ttgcctgggc cttggacgtc
     4861 ttggccaaac gcctccgtcc catgcacgtc tttatcctgg attacgacca atcgcccgcc
     4921 ggctgccggg acgccctgct gcaacttacc tccgggatga tccagaccca cgtcaccacc
     4981 ccaggctcca taccgacgat ctgcgacctg gcgcgcacgt ttgcgcggga gatgggggag
     5041 gctaactgac ccggggcatg cgcggccgca acttgtttat tgcagcttat aatggttaca
     5101 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     5161 gtggtttgtc caaactcatc aatgtatctt atcatgtctg caattgataa cttcgtatag
     5221 catacattat acgaagttat atcgattgtt aactctagat atgggctaaa agaatactgg
     5281 cgccaatatt aagccaaaat ctgtaatatg tctacaattc ttggttgagg tagagagtgg
     5341 ctgtttgcaa ttaaatagta cccagaggca taacactttc tggctagatt ttggttaagg
     5401 caaacttttg tgatatgtaa ccaaatccac ttcactatta aaggctgtac ttgtgatatg
     5461 agaagtgatc agctgtggta gacagttggt tctgtactac tgcatccaga cttgtgttgg
     5521 ttgtatatgt attatagtac ttagttttca ttttaactta ctttctaagg aacactggaa
     5581 ctgcccaatt taatttcaaa agttggagga ttttgttttg agacaagatt tccctctgta
     5641 agcctgttct gtcctcagct ccggagtgct agggctctag gcgtgagcca tcatgctgaa
     5701 tttagattag catttaaaca gaaattatta aagcatagga aagaagtctc agtggttaca
     5761 tttaaatgtc gacctgcagg cgtacgacta gtaaaagagt cgaataaggg cgacacactg
     5821 tcattagcaa ctccttgtcc ttcgatctcg tcaacaacag cttgcagttc aaatacaaga
     5881 cccagaaggc gactattctg gaagcgagct tgaagccaga cgctgagtac gaaaaggggc
     5941 ccgagcttaa gactggccgt cgttttacaa cacagaaaga gtttgtagaa acgcaaaaag
     6001 gccatccgtc aggggccttc tgcttagttt gatgcctggc agttccctac tctcgccttc
     6061 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     6121 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     6181 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     6241 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     6301 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     6361 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     6421 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     6481 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     6541 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     6601 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtgggctaa
     6661 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     6721 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     6781 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     6841 cttttctacg gggtctgacg ctcagtggaa cgacgcgcgc gtaactcacg ttaagggatt
     6901 ttggtcatga gcttgcgccg tcccgtcaag tcagcgtaat gctctgctta gaaaaactca
     6961 tcgagcatca aatgaaattg caatttattc atatcaggat tatcaatacc atatttttga
     7021 aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga
     7081 tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc
     7141 tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag
     7201 aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg
     7261 tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgagg
     7321 cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgagtg caaccggcgc
     7381 aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc
     7441 tggaacgctg tttttccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg
     7501 ataaaatgct tgatggtcgg aagtggcata aattccgtca gccagtttag tctgaccatc
     7561 tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca
     7621 tcgggcttcc catacaagcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc
     7681 catttatacc catataaatc agcatccatg ttggaattta atcgcggcct cgacgtttcc
     7741 cgttgaatat ggctcatatt cttccttttt caatattatt gaagcattta tcagggttat
     7801 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggtcagt
     7861 gttacaacca attaaccaat tctga
//
