LOCUS       sequence-358527-        7287 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Factor IX donor for twinPE and Bxb1 mediated insertion at ALB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7287)
  AUTHORS   Anzalone AV, Gao XD, Podracky CJ, Nelson AT, Koblan LW, Raguram A, 
            Levy JM, Mercer JAM, Liu DR
  TITLE     Programmable deletion, replacement, integration and inversion of 
            large DNA sequences with twin prime editing.
  JOURNAL   Nat Biotechnol. 2022 May;40(5):731-740. doi: 
            10.1038/s41587-021-01133-w. Epub 2021 Dec 9.
  PUBMED    34887556
REFERENCE   2  (bases 1 to 7287)
  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"; doi: 
            "10.1038/s41587-021-01133-w"; journalName: "Nat Biotechnol"; date: 
            "2022-05"; volume: "40"; issue: "5"; pages: "731-740"
COMMENT     Sequence Label: Factor IX donor
FEATURES             Location/Qualifiers
     source          1..7287
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(13..30)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    51..84
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     protein_bind    110..157
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attP site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     polyA_signal    2919..3143
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(3288..4082)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(3288..4082)
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3399..3418)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4009..4028
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(4446..4463)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      4616..4702
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4794..4821
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      4955..5543
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5444..5463
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5697..5714
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5729..5869
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5795..5814)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5955..5977
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 cagataaact caatgatgat gatgatgatg gtcgagactc agcggccgcg gtgccagggc
       61 gtgcccttgg gctccccggg cgcgactagt gaattcagat ctgatatcag gtttgtctgg
      121 tcaaccaccg cggtctcagt ggtgtacggt acaaaccttt tcttaagaga tgtaaaattt
      181 tcatgatgtt ttcttttttg ctaaaactaa agaattattc ttttacattt cagtttttct
      241 tgatcatgaa aacgccaaca aaattctgaa tcggccaaag aggtataatt caggtaaatt
      301 ggaagagttt gttcaaggga accttgagag agaatgtatg gaagaaaagt gtagttttga
      361 agaagcacga gaagtttttg aaaacactga aagaacaact gaattttgga agcagtatgt
      421 tgatggagat cagtgtgagt ccaatccatg tttaaatggc ggcagttgca aggatgacat
      481 taattcctat gaatgttggt gtccctttgg atttgaagga aagaactgtg aattagatgt
      541 aacatgtaac attaagaatg gcagatgcga gcagttttgt aaaaatagtg ctgataacaa
      601 ggtggtttgc tcctgtactg agggatatcg acttgcagaa aaccagaagt cctgtgaacc
      661 agcagtgcca tttccatgtg gaagagtttc tgtttcacaa acttctaagc tcacccgtgc
      721 tgagactgtt tttcctgatg tggactatgt aaattctact gaagctgaaa ccattttgga
      781 taacatcact caaagcaccc aatcatttaa tgacttcact cgggttgttg gtggagaaga
      841 tgccaaacca ggtcaattcc cttggcaggt tgttttgaat ggtaaagttg atgcattctg
      901 tggaggctct atcgttaatg aaaaatggat tgtaactgct gcccactgtg ttgaaactgg
      961 tgttaaaatt acagttgtcg caggtgaaca taatattgag gagacagaac atacagagca
     1021 aaagcgaaat gtgattcgaa ttattcctca ccacaactac aatgcagcta ttaataagta
     1081 caaccatgac attgcccttc tggaactgga cgaaccctta gtgctaaaca gctacgttac
     1141 acctatttgc attgctgaca aggaatacac gaacatcttc ctcaaatttg gatctggcta
     1201 tgtaagtggc tggggaagag tcttccacaa agggagatca gctttagttc ttcagtacct
     1261 tagagttcca cttgttgacc gagccacatg tcttcgatct acaaagttca ccatctataa
     1321 caacatgttc tgtgctggct tccatgaagg aggtagagat tcatgtcaag gagatagtgg
     1381 gggaccccat gttactgaag tggaagggac cagtttctta actggaatta ttagctgggg
     1441 tgaagagtgt gcaatgaaag gcaaatatgg aatatatacc aaggtatccc ggtatgtcaa
     1501 ctggattaag gaaaaaacaa agctcactta atgaaagatg gatttccaag gttaattcat
     1561 tggaattgaa aattaacagg gcctctcact aactaatcac tttcccatct tttgttagat
     1621 ttgaatatat acattctatg atcattgctt tttctcttta caggggagaa tttcatattt
     1681 tacctgagca aattgattag aaaatggaac cactagagga atataatgtg ttaggaaatt
     1741 acagtcattt ctaagggccc agcccttgac aaaattgtga agttaaattc tccactctgt
     1801 ccatcagata ctatggttct ccactatggc aactaactca ctcaattttc cctccttagc
     1861 agcattccat cttcccgatc ttctttgctt ctccaaccaa aacatcaatg tttattagtt
     1921 ctgtatacag tacaggatct ttggtctact ctatcacaag gccagtacca cactcatgaa
     1981 gaaagaacac aggagtagct gagaggctaa aactcatcaa aaacactact ccttttcctc
     2041 taccctattc ctcaatcttt taccttttcc aaatcccaat ccccaaatca gtttttctct
     2101 ttcttactcc ctctctccct tttaccctcc atggtcgtta aaggagagat ggggagcatc
     2161 attctgttat acttctgtac acagttatac atgtctatca aacccagact tgcttccgta
     2221 gtggagactt gcttttcaga acatagggat gaagtaaggt gcctgaaaag tttgggggaa
     2281 aagtttcttt cagagagtta agttatttta tatatataat atatatataa aatatataat
     2341 atacaatata aatatatagt gtgtgtgtat gcgtgtgtgt agacacacac gcatacacac
     2401 atataatgga agcaataagc cattctaaga gcttgtatgg ttatggaggt ctgactaggc
     2461 atgatttcac gaaggcaaga ttggcatatc attgtaacta aaaaagctga cattgaccca
     2521 gacatattgt actctttcta aaaataataa taataatgct aacagaaaga agagaaccgt
     2581 tcgtttgcaa tctacagcta gtagagactt tgaggaagaa ttcaacagtg tgtcttcagc
     2641 agtgttcaga gccaagcaag aagttgaagt tgcctagacc agaggacata agtatcatgt
     2701 ctcctttaac tagcataccc cgaagtggag aagggtgcag caggctcaaa ggcataagtc
     2761 attccaatca gccaactaag ttgtcctttt ctggtttcgt gttcaccatg gaacattttg
     2821 attatagtta atccttctat cttgaatctt ctagagagtt gctgaccaac tgacgtatgt
     2881 ttccctttgt gaattaataa actggtgttc tggttcatct gtgccttcta gttgccagcc
     2941 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     3001 cctttcctaa taaaatgaga aaattgcatc gcattgtctg agtaggtgtc attctattct
     3061 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     3121 tggggatgcg gtgggctcta tgggtcgacc catgggggcc cgccccaact ggggtaacct
     3181 ttgagttctc tcagttgggg gtaatcagca tcatgatgtg gtaccacatc atgatgctga
     3241 ttataagaat gcggccgcca cactctagtg gatctcgagt taataattca gaagaactcg
     3301 tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg
     3361 aggaagcggt cagcccattc gccgccaagc tcttcagcaa tatcacgggt agccaacgct
     3421 atgtcctgat agcggtccgc cacacccagc cggccacagt cgatgaatcc agaaaagcgg
     3481 ccattttcca ccatgatatt cggcaagcag gcatcgccat gggtcacgac gagatcctcg
     3541 ccgtcgggca tgctcgcctt gagcctggcg aacagttcgg ctggcgcgag cccctgatgc
     3601 tcttcgtcca gatcatcctg atcgacaaga ccggcttcca tccgagtacg tgctcgctcg
     3661 atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt atgcagccgc
     3721 cgcattgcat cagccatgat ggatactttc tcggcaggag caaggtgaga tgacaggaga
     3781 tcctgccccg gcacttcgcc caatagcagc cagtcccttc ccgcttcagt gacaacgtcg
     3841 agcacagctg cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc tgcctcgtct
     3901 tgcagttcat tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc
     3961 gctgacagcc ggaacacggc ggcatcagag cagccgattg tctgttgtgc ccagtcatag
     4021 ccgaatagcc tctccaccca agcggccgga gaacctgcgt gcaatccatc ttgttcaatc
     4081 atgcgaaacg atcctcatcc tgtctcttga tcagagcttg atcccctgcg ccatcagatc
     4141 cttggcggca agaaagccat ccagtttact ttgcagggct tcccaacctt accagagggc
     4201 gccccagctg gcaattccgg ttcgcttgct gtccataaaa ccgcccagtc tagctatcgc
     4261 catgtaagcc cactgcaagc tacctgcttt ctctttgcgc ttgcgttttc ccttgtccag
     4321 atagcccagt agctgacatt catccggggt cagcaccgtt tctgcggact ggctttctac
     4381 gcgctcgagg gggggccaaa cggtctccag cttggctgtt ttggcggatg agagaagatt
     4441 ttcagcctga tacagattaa atcagaacgc agaagcggtc tgataaaaca gaatttgcct
     4501 ggcggcagta gcgcggtggt cccacctgac cccatgccga actcagaagt gaaacgccgt
     4561 agcgccgatg gtagtgtggg gtctccccat gcgagagtag ggaactgcca ggcatcaaat
     4621 aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa
     4681 cgctctcctg agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc
     4741 cggagggtgg cgggcaggac gcccgccata aactgccagg catcaaatta agcagaaggc
     4801 catcctgacg gatggccttt ttgcgtttct acaaactctt ttgtttattt ttctaaatac
     4861 attcaaatat gtatccgctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4921 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4981 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     5041 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     5101 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     5161 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     5221 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     5281 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     5341 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     5401 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     5461 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     5521 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     5581 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     5641 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     5701 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     5761 gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     5821 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     5881 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     5941 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     6001 cgcgaggcag cagatcaatt cgcgcgcgaa ggcgaagcgg catgcataat gtgcctgtca
     6061 aatggacgaa gcagggattc tgcaaaccct atgctactcc gtcaagccgt caattgtctg
     6121 attcgttacc aattatgaca acttgacggc tacatcattc actttttctt cacaaccggc
     6181 acggaactcg ctcgggctgg ccccggtgca ttttttaaat acccgcgaga aatagagttg
     6241 atcgtcaaaa ccaacattgc gaccgacggt ggcgataggc atccgggtgg tgctcaaaag
     6301 cagcttcgcc tggctgatac gttggtcctc gcgccagctt aagacgctaa tccctaactg
     6361 ctggcggaaa agatgtgaca gacgcgacgg cgacaagcaa acatgctgtg cgacgctggc
     6421 gattcttctc tcatccgcca aaacagccaa gctggagacc gtttgacatt accctgttat
     6481 ccctagatac attaccctgt tatcccagat gacataccct gttatcccta gatgacatta
     6541 ccctgttatc ccagatgaca ttaccctgtt atccctagat acattaccct gttatcccag
     6601 atgacatacc ctgttatccc tagatgacat taccctgtta tcccagatga cattaccctg
     6661 ttatccctag atacattacc ctgttatccc agatgacata ccctgttatc cctagatgac
     6721 attaccctgt tatcccagat gacattaccc tgttatccct agatacatta ccctgttatc
     6781 ccagatgaca taccctgtta tccctagatg acattaccct gttatcccag atgacattac
     6841 cctgttatcc ctagatacat taccctgtta tcccagatga cataccctgt tatccctaga
     6901 tgacattacc ctgttatccc agatgacatt accctgttat ccctagatac attaccctgt
     6961 tatcccagat gacataccct gttatcccta gatgacatta ccctgttatc ccagatgaca
     7021 ttaccctgtt atccctagat acattaccct gttatcccag atgacatacc ctgttatccc
     7081 tagatgacat taccctgtta tcccagatga cattaccctg ttatccctag atacattacc
     7141 ctgttatccc agatgacata ccctgttatc cctagatgac attaccctgt tatcccagat
     7201 gacattaccc tgttatccct agatacatta ccctgttatc ccagatgaca taccctgtta
     7261 tccctagatg acattaccct gttatcc
//
