LOCUS       sequence-354295-        8373 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targets Rogi1 genomic region with HDR and expresses LAMB3-T2A-GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8373)
  AUTHORS   Aznauryan E, Yermanos A, Kinzina E, Devaux A, Kapetanovic E, 
            Milanova D, Church GM, Reddy ST
  TITLE     Discovery and validation of human genomic safe harbor sites for gene
            and cell therapies.
  JOURNAL   Cell Rep Methods. 2022 Jan 14;2(1):100154. doi: 
            10.1016/j.crmeth.2021.100154. eCollection 2022 Jan 24.
  PUBMED    35474867
REFERENCE   2  (bases 1 to 8373)
  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: "Cell Rep 
            Methods."; date: "2022-01-14"; volume: "2(1):100154. doi"; pages: " 
            10.1016/j.crmeth.2021.100154. eCollection 2022 Jan 24"
COMMENT     Sequence Label: Rogi1-CMV-LAMB3-T2A-GFP-bGH
FEATURES             Location/Qualifiers
     source          1..8373
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1576..1629
                     /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(1633..1635,1636..1638,1639..2352)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1633..1635,1636..1638,1639..2349)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1678..1699)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1939..1958)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2286..2307
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2422..2442)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    complement(3096..3195)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3252..3268)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3252..3268)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3265..3287)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(3440..4028)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3520..3539)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4164..4979)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(4164..4979)
                     /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="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4887..4906
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4971..4990
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     5100..5116
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5100..5116
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     terminator      5117..5163
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     5277..5299
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5291..5308
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5292..5308
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5361..5460
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     enhancer        5761..6140
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6141..6344
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6295..6315
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     6389..6408
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6389..6407
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gtggctcagg tggccagtgc catcctctcc ctcaggcgaa ctctccaggg cctgcagctg
       61 gatctgcccc tggaggagga gacgttgtcc cttccgagag acctggagag tcttgacaga
      121 agcttcaatg gtctccttac tatgtatcag aggaagaggg agcagtttga aaaaataagc
      181 agtgctgatc cttcaggagc cttccggatg ctgagcacag cctacgagca gtcagcccag
      241 gctgctcagc aggtctccga cagctcgcgc cttttggacc agctcaggga cagccggaga
      301 gaggcagaga ggctggtgcg gcaggcggga ggaggaggag gcaccggcag ccccaagctt
      361 gtggccctga ggctggagat gtcttcgttg cctgacctga cacccacctt caacaagctc
      421 tgtggcaact ccaggcagat ggcttgcacc ccaatatcat gccctggtga gctatgtccc
      481 caagacaatg gcacagcctg tggctcccgc tgcaggggtg tccttcccag ggccggtggg
      541 gccttcttga tggcggggca ggtggctgag cagctgcggg gcttcaatgc ccagctccag
      601 cggaccaggc agatgattag ggcagccgag gaatctgcct cacagattca atccagtgcc
      661 cagcgcttgg agacccaggt gagcgccagc cgctcccaga tggaggaaga tgtcagacgc
      721 acacggctcc taatccagca ggtccgggac ttcctaacag accccgacac tgatgcagcc
      781 actatccagg aggtcagcga ggccgtgctg gccctgtggc tgcccacaga ctcagctact
      841 gttctgcaga agatgaatga gatccaggcc attgcagcca ggctccccaa cgtggacttg
      901 gtgctgtccc agaccaagca ggacattgcg cgtgcccgcc ggttgcaggc tgaggctgag
      961 gaagccagga gccgagccca tgcagtggag ggccaggtgg aagatgtggt tgggaacctg
     1021 cggcagggga cagtggcact gcaggaagct caggacacca tgcaaggcac cagccgctcc
     1081 cttcggctta tccaggacag ggttgctgag gttcagcagg tactgcggcc agcagaaaag
     1141 ctggtgacaa gcatgaccaa gcagctgggt gacttctgga cacggatgga ggagctccgc
     1201 caccaagccc ggcagcaggg ggcagaggca gtccaggccc agcagcttgc ggaaggtgcc
     1261 agcgagcagg cattgagtgc ccaagaggga tttgagagaa taaaacaaaa gtatgctgag
     1321 ttgaaggacc ggttgggtca gagttccatg ctgggtgagc agggtgcccg gatccagagt
     1381 gtgaagacag aggcagagga gctgtttggg gagaccatgg agatgatgga caggatgaaa
     1441 gacatggagt tggagctgct gcggggcagc caggccatca tgctgcgctc ggcggacctg
     1501 acaggactgg agaagcgtgt ggagcagatc cgtgaccaca tcaatgggcg cgtgctctac
     1561 tatgccacct gcaaggaggg cagaggaagt cttctaacat gcggtgacgt ggaggagaat
     1621 cccggcccta gcatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     1681 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
     1741 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     1801 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
     1861 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     1921 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     1981 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     2041 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     2101 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     2161 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     2221 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     2281 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     2341 ctgtacaagt aaagcggact agtctagcaa tcaacctctg gattacaaaa tttgtgaaag
     2401 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
     2461 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
     2521 ctggttagtt cttgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
     2581 ggctcggctg ttgggcactg acaattccgt ggtgtttatt tgtgaaattt gtgatgctat
     2641 tgctttattt gtaaccatct agctttattt gtgaaatttg tgatgctatt gctttatttg
     2701 taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc
     2761 aggttcaggg ggagatgtgg gaggtttttt aaagccatgg cactaggact aaaggttggc
     2821 caaagtacaa gatatttgtc ttatctgatg acaactctgt gtcctggact ctcttccaga
     2881 ataagacctt tcctgcagca ctgcttgaac tcctcttagc aagagggaaa catgtgaaat
     2941 gctaccaaaa tagaatagaa gtaaattctt attatattcc tttgttcact catatcctga
     3001 agtgcatcaa atcaggtttt ctcacctgta taatgctgta ttttacttga gttggaataa
     3061 ttttgcttag aaataaataa gtaaaacagc acctgaccca gctttcttgt acaaagttgg
     3121 cattataaga aagcattgct tatcaatttg ttgcaacgaa caggtcacta tcagtcaaaa
     3181 taaaatcatt atttgaggct aggtggaggc tcagtgatga taagtctgcg atggtggatg
     3241 catgtgtcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcag agggcacaat
     3301 cctattccgc gctatccgac aatctccaag acattaggtg gagttcagtt cggcgtatgg
     3361 catatgtcgc tggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     3421 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     3481 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     3541 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     3601 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     3661 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     3721 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc tgtaagacac gacttatcgc
     3781 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     3841 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     3901 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     3961 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     4021 gatctcaaga agatcctttg atcttttcta cggggtctga cgctctattc aacaaagccg
     4081 ccgtcccgtc aagtcagcgt aaatgggtag ggggcttcaa atcgtccgct ctgccagtgt
     4141 tacaaccaat taacaaattc tgattagaaa aactcatcga gcatcaaatg aaactgcaat
     4201 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga
     4261 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
     4321 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
     4381 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagctt atgcatttct
     4441 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc
     4501 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa
     4561 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
     4621 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc
     4681 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
     4741 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg
     4801 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag
     4861 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
     4921 tccatgttgg aatttaatcg cggcctcgag caagacgttt cccgttgaat atggctcata
     4981 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga tgatatattt
     5041 ttatcttgtg caatgtaaca tcagagattt tgagacacaa cgtggctttc ccccgccgct
     5101 ctagaactag tggatccaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt
     5161 tatctgttgt ttgtcgcatt atacgagacg tccaggttgg gatacctgaa acaaaaccca
     5221 tcgtacggcc aaggaagtct ccaataactg tgatccacca caagcgccag ggttttccca
     5281 gtcacgacgt tgtaaaacga cggccagtca tgcataatcc gcacgcatct ggaataagga
     5341 agtgccattc cgcctgacct caaataatga ttttattttg actgatagtg acctgttcgt
     5401 tgcaacagat tgatgagcaa tgctttttta taatgccaac tttgtacaaa aaagcaggct
     5461 ctgcatttaa gtaggattca ataattttaa agtgcaggga caaaatttcc tcatatggct
     5521 cactagctac attgcaaatt tcttgaaatc agaacacaga agtgcagtcc tgtgctcgca
     5581 atgcagactt gcagggtgta gaggcataaa tggctccaga gccagggaca tgggtccaga
     5641 ggggggtagt ctccagaaga ctcctttcgg gcctattacc atgcctcaga ggtccaagtg
     5701 gggcatggtg aatatattat cctttatatt atatttctta tatgtctaca actgccactt
     5761 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
     5821 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
     5881 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
     5941 ctttccattg acgtcaatgg gtggactatt tacggtaaac tgcccacttg gcagtacatc
     6001 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
     6061 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
     6121 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
     6181 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
     6241 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
     6301 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
     6361 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
     6421 gcggccgcca ccatgagacc attcttcctc ttgtgttttg ccctgcctgg cctcctgcat
     6481 gcccaacaag cctgctcccg tggggcctgc tatccacctg ttggggacct gcttgttggg
     6541 aggacccggt ttctccgagc ttcatctacc tgtggactga ccaagcctga gacctactgc
     6601 acccagtatg gcgagtggca gatgaaatgc tgcaagtgtg actccaggca gcctcacaac
     6661 tactacagtc accgagtaga gaatgtggct tcatcctccg gccccatgcg ctggtggcag
     6721 tcccagaatg atgtgaaccc tgtctctctg cagctggacc tggacaggag attccagctt
     6781 caagaagtca tgatggagtt ccaggggccc atgcctgccg gcatgctgat tgagcgctcc
     6841 tcagacttcg gtaagacctg gcgagtgtac cagtacctgg ctgccgactg cacctccacc
     6901 ttccctcggg tccgccaggg tcggcctcag agctggcagg atgttcggtg ccagtccctg
     6961 cctcagaggc ctaatgcacg cctaaatggg gggaaggtcc aacttaacct tatggattta
     7021 gtgtctggga ttccagcaac tcaaagtcaa aaaattcaag aggtggggga gatcacaaac
     7081 ttgagagtca atttcaccag gctggcccct gtgccccaaa ggggctacca ccctcccagc
     7141 gcctactatg ctgtgtccca gctccgtctg caggggagct gcttctgtca cggccatgct
     7201 gatcgctgcg cacccaagcc tggggcctct gcaggcccct ccaccgctgt gcaggtccac
     7261 gatgtctgtg tctgccagca caacactgcc ggcccaaatt gtgagcgctg tgcacccttc
     7321 tacaacaacc ggccctggag accggcggag ggccaggacg cccatgaatg ccaaaggtgc
     7381 gactgcaatg ggcactcaga gacatgtcac tttgaccccg ctgtgtttgc cgccagccag
     7441 ggggcatatg gaggtgtgtg tgacaattgc cgggaccaca ccgaaggcaa gaactgtgag
     7501 cggtgtcagc tgcactattt ccggaaccgg cgcccgggag cttccattca ggagacctgc
     7561 atctcctgcg agtgtgatcc ggatggggca gtgccagggg ctccctgtga cccagtgacc
     7621 gggcagtgtg tgtgcaagga gcatgtgcag ggagagcgct gtgacctatg caagccgggc
     7681 ttcactggac tcacctacgc caacccgcag ggctgccacc gctgtgactg caacatcctg
     7741 gggtcccgga gggacatgcc gtgtgacgag gagagtgggc gctgcctttg tctgcccaac
     7801 gtggtgggtc ccaaatgtga ccagtgtgct ccctaccact ggaagctggc cagtggccag
     7861 ggctgtgaac cgtgtgcctg cgacccgcac aactccctca gcccacagtg caaccagttc
     7921 acagggcagt gcccctgtcg ggaaggcttt ggtggcctga tgtgcagcgc tgcagccatc
     7981 cgccagtgtc cagaccggac ctatggagac gtggccacag gatgccgagc ctgtgactgt
     8041 gatttccggg gaacagaggg cccgggctgc gacaaggcat caggccgctg cctctgccgc
     8101 cctggcttga ccgggccccg ctgtgaccag tgccagcgag gctactgcaa tcgctacccg
     8161 gtgtgcgtgg cctgccaccc ttgcttccag acctatgatg cggacctccg ggagcaggcc
     8221 ctgcgctttg gtagactccg caatgccacc gccagcctgt ggtcagggcc tgggctggag
     8281 gaccgtggcc tggcctcccg gatcctagat gcaaagagta agattgagca gatccgagca
     8341 gttctcagca gccccgcagt cacagagcag gag
//
