LOCUS       sequence-403013-        3711 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For production of template RNA for use in PRINT, GFP under CMV 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3711)
  AUTHORS   Zhang X, Van Treeck B, Horton CA, McIntyre JJR, Palm SM, Shumate JL,
            Collins K
  TITLE     Harnessing eukaryotic retroelement proteins for transgene insertion 
            into human safe-harbor loci.
  JOURNAL   Nat Biotechnol. 2024 Feb 20. doi: 10.1038/s41587-024-02137-y.
  PUBMED    38379101
REFERENCE   2  (bases 1 to 3711)
  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 
            Biotechnol. 2024 Feb 20. doi: 10.1038/s41587-024-02137-y."
COMMENT     Sequence Label: TCARZ-CMV-GFP-PAmin-GF3-R4A22
FEATURES             Location/Qualifiers
     source          1..3711
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(171..962,963..1031))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     794..813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1032..1136)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1188..1207
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1188..1206
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     enhancer        1321..1624
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1625..1828
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1779..1799
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1949..1958
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1955..1957,1958..1960,1961..2674)
                     /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(1955..1957,1958..1960,1961..2671)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2000..2021)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2261..2280)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2608..2629
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     rep_origin      complement(3123..3711)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3203..3222)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      301 atgataccgc gagatccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
      721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1141 tttccccgaa aagtgccacc tgacgtccca atgatcttaa ggaattctaa tacgactcac
     1201 tatagggtaa acggcgggag taactatgac tctcttaagg agtcatagag ccagaacctc
     1261 ctcgtggtcc cgctgggcac agggattaat ttttctgtgg caaatttgac tggctctaga
     1321 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
     1381 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
     1441 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
     1501 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
     1561 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
     1621 catggtgatg cggttttggc agtacatcaa tgggcgtgga taggggtttg actcacgggg
     1681 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
     1741 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
     1801 acggtgggag gtctatataa gcagagctcg tttagtgaac cgcttgtctc gctccgggga
     1861 acgctcggaa actcccggcc gccgccaccc gcgtctgttc tgttacacaa gggaagaaaa
     1921 gccgctgccg cactccgagt gtcgtacggc caccatggtg agcaagggcg aggagctgtt
     1981 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     2041 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     2101 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
     2161 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     2221 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     2281 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     2341 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     2401 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
     2461 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     2521 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag
     2581 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     2641 gatcactctc ggcatggacg agctgtacaa gtaaaataaa agatctttat gttcattaga
     2701 tctgtgtgtt ggtttttgtg tggctgaggt agataatctt tgtatagtgg ggggggatct
     2761 catgtaccgg gtttctttta tttgattttc aataaaacag acggtagcta ggttcgcaag
     2821 gcagccacaa gccaaagata ggtagggtgc tcatagtgag tagggacagt gccttttgat
     2881 tcacaacgcg tcaataccat ctgacacgga tacccttacc ggacttgtca tgatctccca
     2941 gacttgtcca aggtggacgg gccaccttta cttaacccgg aaaaggaaca tatattaatt
     3001 atatgtgttc ggaaaatagc aaaaaaaaaa aaaaaaaaaa aaaaagtctt catcggaaag
     3061 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     3121 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     3181 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     3241 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     3301 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     3361 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     3421 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     3481 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     3541 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     3601 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     3661 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca a
//
