LOCUS       sequence-474300-        5877 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For production of C-term flag-tagged TaGu EN-tuned ORF mRNA for mRNA
            transfection in PRINT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5877)
  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 5877)
  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: pT7-TaGu-ENT-3xF-ORF
FEATURES             Location/Qualifiers
     source          1..5877
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      117..126
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3684..3749
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     rep_origin      complement(4165..4753)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4245..4264)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4924..5715,5716..5784))
                     /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     5547..5566
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(5785..5877,1..12))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcccaatga ttaatacgac
       61 tcactataag gaataaacta gtattcttct ggtccccaca gactcagaga gaacccgcca
      121 ccatggagaa ggtgatggtc accgttccgg acaagaaccc tccatgtcca tgctgcggca
      181 cccgggtgaa cagcgtgctg aaccttatcg agcacctgaa ggtcagccac ggaaagcggg
      241 gcgtgtgctt ccggtgcgcg aagtgtggga aggaaaatag caactaccac agcgtggtgt
      301 gccatttccc aaaatgtaga ggacctgaga cagagaaagc tccagccggc gagtggattt
      361 gtgaagtgtg caacagagac ttcaccacca aaatcggcct gggccagcat aagagattag
      421 ctcaccccgc tgttcggaat caggagagaa tcgtcgccag ccagcctaag gaaactagca
      481 acagaggcgc tcacaaaaga tgctggacca aagaggagga ggaactgctg atcagactgg
      541 aagcccagtt tgagggcaac aagaacatca acaagctgat cgcagaacac atcaccacaa
      601 agaccgccaa gcagatctcc gataagagga gactgctgag ccgcaagcct gccgaggagc
      661 ctagagagga acctggcaca tgccaccaca ccagacgggc agctgccagc ctgagaacag
      721 agcccgagat gtcccaccac gctcaggccg aagatcggga taatggccct ggccggagac
      781 ccctgcctgg tagagccgcc gccggcggca gaacaatgga cgagatcagg agacaccctg
      841 acaagggcaa tggacagcag cggcctacca aacagaagtc tgaggagcag ctgcaagcct
      901 actacaaaaa gacactggag gagcggctga gcgccggagc cctgaatacc ttcccccgcg
      961 ccttcaagca agtgatggaa ggcagagata tcaagctggt gatcaaccag acagcccagg
     1021 actgcttcgg ctgtctggag agcatctctc agatcagaac agcgaccaga gataagaaag
     1081 ataccgtgac cagagagaag cacccgaaga agccattcca aaagtggatg aaggaccggg
     1141 ccataaagaa gggcaactac ctgaggttcc agagattgtt ctacctggac cgaggcaaac
     1201 tggccaagat catcctggac gatatcgaat gcctgagctg cgacatcccc ctgtctgaga
     1261 tctacagcgt gtttaagaca agatgggaaa ccacaggaag cttcaaatct ctgggcgatt
     1321 ttaaaaccta cggcaaggcc gacaacactg ccttcagaga actgattaca gctaaggaaa
     1381 tcgagaaaaa cgtgcaggag atgagcaagg gcagcgcccc tggccctgac ggtatcaccc
     1441 tgggagatgt ggtgaaaatg gaccccgagt tcagccgcac aatggagatc ttcaacctgt
     1501 ggctgaccac tggcaaaatc cctgacatgg tgcggggatg ccgcaccgtg ctgataccta
     1561 agagcagcaa gccagataga ctgaaggata tcaacaactg gcggcctatc acaatcggca
     1621 gcatcctgct ccggctgttt agcagaatcg taaccgcccg tctgagtaag gcctgtcctc
     1681 tgaaccccag acagcggggc ttcatccggg ccgccggctg cagcgagaac ctcaaactgc
     1741 tgcagaccat catctggtcg gccaaacgag agcacagacc tctcggcgtg gtgttcgtgg
     1801 acatcgccaa agcttttgat accgtctccc accagcacat catccacgcc ctgcagcagc
     1861 gggaagtgga ccctcacatc gtgggactgg tgtccaacat gtacgagaac atcagcactt
     1921 atatcaccac caagagaaac acccacacag acaagatcca gatcagagtg ggcgttaagc
     1981 agggcgatcc tatgtctcct ctgctgttca acctggctat ggaccctctg ctctgtaaac
     2041 tggaggagtc tggcaaggga taccacagag gccaaagctc catcaccgct atggcctttg
     2101 ccgacgacct ggtgctgctg tctgattctt gggagaacat gaataccaac ataagcatcc
     2161 tggaaacatt ctgcaacctt acaggcctga aaacccaggg acaaaagtgc cacggctttt
     2221 acatcaagcc taccaaagac agctacacca tcaacgactg cgccgcctgg accatcaacg
     2281 gcaccccttt aaacatgatc gaccctggag aaagcgagaa gtatctgggc ctgcagttcg
     2341 acccctggat cggcatcgct agaagcggcc tgagcacaaa gctggacttt tggctgcaga
     2401 gaattgacca ggcccctctg aagcctctgc agaaaaccga catcctgaaa acctacacaa
     2461 tccctcggct gatctacatt gccgatcaca gcgaggtaaa gaccgccctg ctggaaaccc
     2521 tggaccaaaa gatcagaacc gccgtgaagg aatggctcca cctgcccccc tgcacctgcg
     2581 acgctatcct gtacagcagc accagggacg gcggcctggg catcaccaag ctggcgggcc
     2641 tgatcccctc cgtccaggct agacggctgc atagaatcgc ccagagcagc gatgacacaa
     2701 tgaagtgctt tatggaaaag gaaaagatgg aacagctgca caagaagctg tggattcagg
     2761 ccggtggcga cagagagaac attcctagca tctgggaggc gccgcctagt agcgagcctc
     2821 ccaacaacgt gtctaccaat tctgagtggg aagcccctac acagaaggac aagttcccta
     2881 agccttgtaa ttggagaaag aacgagttca agaagtggac aaagctggcc tctcagggcc
     2941 ggggaattgt gaatttcgag cgggacaaga tcagcaatca ctggattcag tactacagaa
     3001 gaatcccaca cagaaagctg ctgacggccc ttcagctgag agccaacgtg taccccacgc
     3061 gggagttcct ggccagaggt agacaggacc agtacatcaa ggcctgcaga cattgtgatg
     3121 ctgatatcga gtcttgcgcc cacatcatcg gcaactgccc tgtgacacag gacgcgagaa
     3181 tcaaaagaca caactacatc tgcgagctgc tgctggaaga ggccaagaag aaggactggg
     3241 tggtgttcaa ggaaccccac atcagagaca gcaataagga actctataaa cctgacctga
     3301 tcttcgtgaa ggacgcccgg gccctggtcg tggacgtgac cgtgagatac gaggccgcca
     3361 agtctagcct ggaggaggct gccgccgaga aagtgcggaa gtacaagcac cttgaaacag
     3421 aagtgcggca cctgaccaac gccaaggacg tcacattcgt gggcttcccc ctgggcgccg
     3481 caggcaaatg gcaccaggat aacttcaagc tgctgacaga gctgggcctg agcaagtccc
     3541 gccaagtgaa gatggccgag accttcagca ccgtggccct gttctcctct gtggacatcg
     3601 tgcacatgtt cgctagcaga gccagaaaga gcatggtgat gcatatgggt ggaggtagcg
     3661 ggggcagtgg agggatgggg agcgactaca aagaccatga cggtgattat aaagatcatg
     3721 acatcgatta caaggatgac gatgacaaga agtgatgacc tcgagctggt actgcatgca
     3781 cgcaatgcta gctgcccctt tcccgtcctg ggtaccccga gtctcccccg acctcgggtc
     3841 ccaggtatgc tcccacctcc acctgcccca ctcaccacct ctgctagttc cagacacctc
     3901 ccaagcacgc agcaatgcag ctcaaaacgc ttagcctagc cacaccccca cgggaaacag
     3961 cagtgattaa cctttagcaa taaacgaaag tttaactaag ctatactaac cccagggttg
     4021 gtcaatttcg tgccagccac accctggagc tagcaaaaaa aaaaaaaaaa aaaaaaaaaa
     4081 aaaaaaagtc ttcatcggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     4141 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     4201 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     4261 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     4321 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     4381 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     4441 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     4501 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     4561 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     4621 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     4681 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     4741 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     4801 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     4861 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     4921 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     4981 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     5041 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     5101 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     5161 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     5221 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     5281 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     5341 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     5401 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     5461 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     5521 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     5581 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     5641 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     5701 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     5761 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     5821 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaat
//
