LOCUS       sequence-426732-        8867 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of N-flag tagged ZoAl-ENT R2 protein in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8867)
  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 8867)
  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: pcDNA3.1 ZoAl-ENT N-1F (R1103A)
FEATURES             Location/Qualifiers
     source          1..8867
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      917..926
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             926..949
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      4163..4172
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(4458..4475)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4464..4688
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4734..5162
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4821..4840)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5031..5052
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5171..5191)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5176..5505
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5356..5491
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5572..6366
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5626..5645)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6236..6255
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6540..6661
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6577..6596)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6631..6650
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6710..6726)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6710..6726)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6723..6745)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6734..6750
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(6758..6764,6765..6782,6783..6788))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6803..6824
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6941..6958)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7112..7700)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7192..7211)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7871..8662,8663..8731))
                     /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     8494..8513
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8732..8836)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttgcca ccatggatta caaggatgac gacgataagg gtaccatgaa
      961 catcgtgaag gtgaccgtgc ccgacaagaa tcccccttgt ccttgctgtg gcgtgcgcct
     1021 gaacagcgtg ctggcactga tcgagcacct gaagggctcc cacggaagga gaagggtgtg
     1081 cttccgctgt gccaagtgcg gccgggagaa ctttaatcac cacagcaccg tgtgtcacta
     1141 cgccaagtgc aagggcccac agatcgagag accacccgtg ggcgagtgga tctgtgaggt
     1201 gtgcggcagg gatttcacca caaagatcgg cctgggccag cacaagcgcc acatgcacgc
     1261 catggtgcgc aaccaggagc ggatcgacgc cagccagccc aaggagacct ccaatcgcgg
     1321 cgcccacaag cggtgctgga caaaggagga ggaggagctg ctgatgaagc tggaggtgca
     1381 gtttgagaac cacaagaaca tcaataagct gatcgccgag cagctgacca caaagaccgc
     1441 caagcagatc tccgataagc gccggatgct gctgaagaag ggcagaggca ccacaggcaa
     1501 cctggagaca gagcctggca tgagccacca gtcccaggcc aaggtgaagg acaatggact
     1561 gggaggcgat cacctgccag gaggacctgt ggtggacaag ggcaccatcg gcaagccagg
     1621 ccagcacctg gacaccgata attctcacca gatcacagca ggcaagaaga agggaggagg
     1681 actgcaggcc agatacagaa ggcgcatcat gaagaggctg gccgccggca ccatcaacat
     1741 cttcccaaag gtgtttaagg agctgatcaa cgaccaggag gccaggcccc tgatcaatca
     1801 gaccacagag gactgtttcg gcctgctgga ttccgcctgc cagatcagaa ccgccctgag
     1861 ggagaagggc aagtctcagg aggagcggcc cagaaagcag taccagaagt ggatgaagaa
     1921 gcgcgccatc aagcggggcg actatctgcg gttccagaga ctgtttcacc tggatagagg
     1981 caagctggcc aggatcatcc tggacaacac cgagtccctg tcttgtgata tcagcccttc
     2041 cgagatctac agcgtgttca aggcccgctg ggagacacca ggccacttca atggcctggg
     2101 cgattttgag atcaagggca aggccaacaa taaggccttc cgggacttta tcaccgccaa
     2161 ggagatcgag aagaacgtga gagagatgtc taagggcagc gcaccaggac ctgatggaat
     2221 cgccctgggc gacatcaaga agatggaccc cggctattct cggaccgccg agctgtttaa
     2281 tctgtggctg acagccggcg acatcccaga tatggtgagg ggatgcagga ccgtgctgat
     2341 cccaaagagc accacacccg agcgcctgaa ggacatcaac aattggcggc ccatcacaat
     2401 cggctccatc ctgctgagac tgttctctag gatcatcacc gccagaatga caaaggcatg
     2461 tccactgaac ccaaggcaga ggggctttat ctccgcacca ggctgctctg agaacctgaa
     2521 gctgctgcag agcatcatca ggaccgccaa gaatgagcac aagcccctgg gcgtgatctt
     2581 cgtggacatc gccaaggcct ttgatacagt gtcccaccag cacatcatcc acgtgctgca
     2641 gcagcggaga gtggaccccc acatcgtggg cctggtgaac aatatgtaca aggacatctc
     2701 cacctatgtg accacaaaga agaacaccca cacagacaag atccagatca gagtgggcgt
     2761 gaagcagggc gatcctctgt ctccactgct gttcaatctg gccatggacc cactgctgtg
     2821 taagctggag gagagcggca agggcttcca caggggccag agctccatca ccgccatggc
     2881 ctttgccgac gatctggtgc tgctgtctga tagctgggag aacatgaagg agaatatcaa
     2941 gatcctggag acattctgta atctgaccgg cctgaagaca cagggccaga agtgccacgg
     3001 cttttacatc aagcctacca aggactccta tacaatcaac aattgcccag cctggaccat
     3061 caacggcaca cctctgaaca tgatcaatcc aggcgagtct gagaagtacc tgggactgca
     3121 gatcgaccca tggaccggcg tggccaagta tgatctgagc acaaagctga agatctggct
     3181 ggagtccatc gacagggccc ccctgaagcc tctgcagaag ctggatatcc tgaagaccta
     3241 cacaatcccc cgcctgacct atctggccga tcacagcgag atgaaggcag gagcactgga
     3301 ggcactggac cagcagatca ggaccgccgt gaaggattgg ctgcacctgc cttcctgcac
     3361 atgtgacgcc atcctgtacg tgtctaccag ggatggagga ctgggagtga caaagctggc
     3421 aggactgatc ccatctgtgc aggcaaggag gctgcacagg atcgcacaga gccctgacga
     3481 gaccatgaag gatttcctgg agaaggccca gatggagaag atgtacgaga agctgtgggt
     3541 gcaggccggc ggcaagaaga agggcatgcc atccatctgg gaggccctgc ccatgacagt
     3601 gcctccaacc aacacaggca atctgtctga gtgggaggcc ccaaatccca agagcaagta
     3661 tcctaagcca tgcgactggc ggagaaagga gctgaagaag tggaccaagc tggagagcca
     3721 gggcagaggc gtgaagaact ttaggaatga cacaatctcc aacgattgga ttcagtacta
     3781 taggcgcatc ccacaccgca agctgctgac cgccatccag ctgcgcgcca acgtgtaccc
     3841 aacaagggag ttcctggcac ggggcagagg cgataattat gtgaagtttt gtcggcactg
     3901 cgaggccgac ctggagacct gtggccacat catcggcttc tgccccgtga caaaggacgc
     3961 ccggatcaag agacacaaca ggatctgtga tagactgtgc gaggaggcag caaagaggga
     4021 gtgggtggtg tttaaggagc cccacctgag ggacgccacc acagagctgt tcaagcctga
     4081 tgtgatcttt gtgaaggagg acagggcact ggtggtggat gtgaccgtgc ggtacgagtc
     4141 cgccaagacc acactggagg ctgccgccat ggagaaggtg gataagtata agcacctgga
     4201 ggccgaggtg aaggagctga caaatgccaa ggacgtggtg ttcatgggat ttccactggg
     4261 agcagcgggc aagttctaca agggcaactt taatctgctg gagaccctgg gcctgcctaa
     4321 gacaagacag ctgtctgtgg ccaagaccct gagcacctac gccctgatgt ctagcgtgga
     4381 catcgtgcac atgttcgcct ccaggtctcg caagcctaac gtgtagtcta gagggcccgt
     4441 ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     4501 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     4561 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     4621 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     4681 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
     4741 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     4801 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     4861 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     4921 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     4981 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     5041 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     5101 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     5161 ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     5221 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     5281 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     5341 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     5401 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc
     5461 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct
     5521 tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa
     5581 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     5641 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     5701 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     5761 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     5821 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     5881 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     5941 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     6001 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     6061 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat
     6121 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     6181 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     6241 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     6301 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     6361 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     6421 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     6481 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca
     6541 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     6601 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     6661 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     6721 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     6781 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     6841 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     6901 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     6961 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     7021 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     7081 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     7141 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     7201 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     7261 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     7321 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     7381 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     7441 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     7501 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     7561 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     7621 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     7681 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     7741 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     7801 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     7861 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     7921 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     7981 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     8041 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     8101 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     8161 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     8221 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     8281 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     8341 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     8401 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     8461 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     8521 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     8581 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     8641 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     8701 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     8761 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     8821 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
