LOCUS       sequence-477706-        5829 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For production of C-term flag-tagged ZoAl EN-dead ORF mRNA for mRNA 
            transfection in PRINT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5829)
  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 5829)
  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-END-3xF-ORF
FEATURES             Location/Qualifiers
     source          1..5829
                     /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(4117..4705)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4197..4216)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4876..5667,5668..5736))
                     /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     5499..5518
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(5737..5829,1..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 tcaagcctgc tgtgatcttt gtgaaggagg acagggcact ggtggtggct gtgaccgtgc
     3301 ggtacgagtc cgccaagacc acactggagg ctgccgccat ggagaaggtg gataagtata
     3361 agcacctgga ggccgaggtg aaggagctga caaatgccaa ggacgtggtg ttcatgggat
     3421 ttccactggg agcaaggggc 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 aacctttaac aaaaaacgaa aatttaacta agctatacta
     3961 accccagggt tggtcaattt cgtgccagcc acaccctgga gctaaaaaaa aaaaaaaaaa
     4021 aaaaaaaaaa aaaaaaaaag tcttcatcgg aaagaacatg tgagcaaaag gccagcaaaa
     4081 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     4141 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     4201 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     4261 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     4321 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     4381 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     4441 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     4501 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     4561 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     4621 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     4681 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     4741 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     4801 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     4861 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     4921 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     4981 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     5041 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     5101 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     5161 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     5221 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     5281 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     5341 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     5401 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     5461 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     5521 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     5581 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     5641 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     5701 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     5761 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     5821 taaacaaat
//
