LOCUS       sequence-476589-        5828 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For production of C-term flag-tagged ZoAl 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 5828)
  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 5828)
  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-ZoAl-ENT-3xF-ORF
FEATURES             Location/Qualifiers
     source          1..5828
                     /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"
     regulatory      3333..3342
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3636..3701
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     rep_origin      complement(4116..4704)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4196..4215)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4875..5666,5667..5735))
                     /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     5498..5517
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(5736..5828,1..12))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcccaatga ttaatacgac
       61 tcactataag gaataaacta gtattcttct ggtccccaca gactcagaga gaacccgcca
      121 ccatggataa catcgtgaag gtgaccgtgc ccgacaagaa tcccccttgt ccttgctgtg
      181 gcgtgcgcct gaacagcgtg ctggcactga tcgagcacct gaagggctcc cacggaagga
      241 gaagggtgtg cttccgctgt gccaagtgcg gccgggagaa ctttaatcac cacagcaccg
      301 tgtgtcacta cgccaagtgc aagggcccac agatcgagag accacccgtg ggcgagtgga
      361 tctgtgaggt gtgcggcagg gatttcacca caaagatcgg cctgggccag cacaagcgcc
      421 acatgcacgc catggtgcgc aaccaggagc ggatcgacgc cagccagccc aaggagacct
      481 ccaatcgcgg cgcccacaag cggtgctgga caaaggagga ggaggagctg ctgatgaagc
      541 tggaggtgca gtttgagaac cacaagaaca tcaataagct gatcgccgag cagctgacca
      601 caaagaccgc caagcagatc tccgataagc gccggatgct gctgaagaag ggcagaggca
      661 ccacaggcaa cctggagaca gagcctggca tgagccacca gtcccaggcc aaggtgaagg
      721 acaatggact gggaggcgat cacctgccag gaggacctgt ggtggacaag ggcaccatcg
      781 gcaagccagg ccagcacctg gacaccgata attctcacca gatcacagca ggcaagaaga
      841 agggaggagg actgcaggcc agatacagaa ggcgcatcat gaagaggctg gccgccggca
      901 ccatcaacat cttcccaaag gtgtttaagg agctgatcaa cgaccaggag gccaggcccc
      961 tgatcaatca gaccacagag gactgtttcg gcctgctgga ttccgcctgc cagatcagaa
     1021 ccgccctgag ggagaagggc aagtctcagg aggagcggcc cagaaagcag taccagaagt
     1081 ggatgaagaa gcgcgccatc aagcggggcg actatctgcg gttccagaga ctgtttcacc
     1141 tggatagagg caagctggcc aggatcatcc tggacaacac cgagtccctg tcttgtgata
     1201 tcagcccttc cgagatctac agcgtgttca aggcccgctg ggagacacca ggccacttca
     1261 atggcctggg cgattttgag atcaagggca aggccaacaa taaggccttc cgggacttta
     1321 tcaccgccaa ggagatcgag aagaacgtga gagagatgtc taagggcagc gcaccaggac
     1381 ctgatggaat cgccctgggc gacatcaaga agatggaccc cggctattct cggaccgccg
     1441 agctgtttaa tctgtggctg acagccggcg acatcccaga tatggtgagg ggatgcagga
     1501 ccgtgctgat cccaaagagc accacacccg agcgcctgaa ggacatcaac aattggcggc
     1561 ccatcacaat cggctccatc ctgctgagac tgttctctag gatcatcacc gccagaatga
     1621 caaaggcatg tccactgaac ccaaggcaga ggggctttat ctccgcacca ggctgctctg
     1681 agaacctgaa gctgctgcag agcatcatca ggaccgccaa gaatgagcac aagcccctgg
     1741 gcgtgatctt cgtggacatc gccaaggcct ttgatacagt gtcccaccag cacatcatcc
     1801 acgtgctgca gcagcggaga gtggaccccc acatcgtggg cctggtgaac aatatgtaca
     1861 aggacatctc cacctatgtg accacaaaga agaacaccca cacagacaag atccagatca
     1921 gagtgggcgt gaagcagggc gatcctctgt ctccactgct gttcaatctg gccatggacc
     1981 cactgctgtg taagctggag gagagcggca agggcttcca caggggccag agctccatca
     2041 ccgccatggc ctttgccgac gatctggtgc tgctgtctga tagctgggag aacatgaagg
     2101 agaatatcaa gatcctggag acattctgta atctgaccgg cctaaagaca cagggccaga
     2161 agtgccacgg cttttacatc aagcctacca aggactccta tacaatcaac aattgcccag
     2221 cctggaccat caacggcaca cctctgaaca tgatcaatcc aggcgagtct gagaagtacc
     2281 tgggactgca gatcgaccca tggaccggcg tggccaagta tgatctgagc acaaagctga
     2341 agatctggct ggagtccatc gacagggccc ccctgaagcc tctgcagaag ctggatatcc
     2401 tgaaaaccta cacaatcccc cgcctgacct atctggccga tcacagcgag atgaaggcag
     2461 gagcactgga ggcactggac cagcagatca ggaccgccgt gaaggattgg ctgcacctgc
     2521 cttcctgcac atgtgacgcc atcctgtacg tgtctaccag ggatggagga ctgggagtga
     2581 caaagctggc aggactgatc ccatctgtgc aggcaaggag gctgcacagg atcgcacaga
     2641 gccctgacga gaccatgaag gatttcctgg agaaggccca gatggagaag atgtacgaga
     2701 agctgtgggt gcaggccggc ggcaagaaga agggcatgcc atccatctgg gaggccctgc
     2761 ccatgacagt gcctccaacc aacacaggca atctgtctga gtgggaggcc ccaaatccca
     2821 agagcaagta tcctaagcca tgcgactggc ggagaaagga gctgaagaag tggaccaagc
     2881 tggagagcca gggcagaggc gtgaagaact ttaggaatga cacaatctcc aacgattgga
     2941 ttcagtacta taggcgcatc ccacaccgca agctgctgac cgccatccag ctgcgcgcca
     3001 acgtgtaccc aacaagggag ttcctggcac ggggcagagg cgataattat gtgaagtttt
     3061 gtcggcactg cgaggccgac ctggagacct gtggccacat catcggcttc tgccccgtga
     3121 caaaggacgc ccggatcaag agacacaaca ggatctgtga tagactgtgc gaggaggcag
     3181 caaagaggga gtgggtggtg tttaaggagc cccacctgag ggacgccacc acagagctgt
     3241 tcaagcctga tgtgatcttt gtgaaggagg acagggcact ggtggtggat gtgaccgtgc
     3301 ggtacgagtc cgccaagacc acactggagg ctgccgccat ggagaaggtg gataagtata
     3361 agcacctgga ggccgaggtg aaggagctga caaatgccaa ggacgtggtg ttcatgggat
     3421 ttccactggg agcagcgggc aagttctaca agggcaactt taatctgctg gagaccctgg
     3481 gcctgcctaa gacaagacag ctgtctgtgg ccaagaccct gagcacctac gccctgatgt
     3541 ctagcgtgga catcgtgcac atgttcgcct ccaggtctcg caagcctaac gtgcatatgg
     3601 gtggaggtag cgggggcagt ggagggatgg ggagcgacta caaagaccat gacggtgatt
     3661 ataaagatca tgacatcgat tacaaggatg acgatgacaa gaagtgatga cctcgagctg
     3721 gtactgcatg cacgcaatgc tagctgcccc tttcccgtcc tgggtacccc gagtctcccc
     3781 cgacctcggg tcccaggtat gctcccacct ccacctgccc cactcaccac ctctgctagt
     3841 tccagacacc tcccaagcac gcagcaatgc agctcaaaac gcttagccta gccacacccc
     3901 cacgggaaac agcagtgatt aacctttagc aataaacgaa agtttaacta agctatacta
     3961 accccagggt tggtcaattt cgtgccagcc acaccctgga gctagcaaaa aaaaaaaaaa
     4021 aaaaaaaaaa aaaaaaaagt cttcatcgga aagaacatgt gagcaaaagg ccagcaaaag
     4081 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     4141 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     4201 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     4261 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     4321 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     4381 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     4441 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     4501 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     4561 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     4621 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     4681 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     4741 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     4801 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     4861 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     4921 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     4981 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     5041 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     5101 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     5161 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     5221 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     5281 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     5341 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     5401 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     5461 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     5521 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     5581 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     5641 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     5701 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     5761 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     5821 aaacaaat
//
