LOCUS       sequence-479513-        6309 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For targeted gene knock-in at NHP IGH locus and B cell-specific 
            expression of GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6309)
  AUTHORS   Castelli JMP, Poljakov K, Jwa Y, Cassidy ME, Gray MD, Sanchez Gaytan
            JN, Enstrom MR, Linton JD, Rongvaux A, Taylor JJ, Adair JE
  TITLE     In vivo production of an anti-HIV antibody from primate 
            hematopoietic cells by non-viral knock-in.
  JOURNAL   bioRxiv [Preprint]. 2025 May 8:2025.05.02.651933. doi: 
            10.1101/2025.05.02.651933.
  PUBMED    40654608
REFERENCE   2  (bases 1 to 6309)
  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: "bioRxiv 
            [Preprint]. 2025 May 8:2025.05.02.651933. doi: 
            10.1101/2025.05.02.651933."
COMMENT     Sequence Label: pVH4p_eGFP
FEATURES             Location/Qualifiers
     source          1..6309
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      77..665
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     566..585
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   902..1031
                     /label=AAV2 ITR
     CDS             join(2069..2071,2072..2074,2075..2788)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2069..2071,2072..2074,2075..2785)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2114..2135)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2375..2394)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2722..2743
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2801..2935
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2825..2844)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2879..2898
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3490..3619
                     /label=AAV2 ITR
     rep_origin      3990..4503
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4077..4096)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4287..4308
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4985..5004)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5104..5126
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5164..5182)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5250..5354
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5355..5423,5424..6215)
                     /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     complement(5573..5592)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
       61 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
      121 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
      181 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
      241 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
      301 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
      361 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
      421 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
      481 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
      541 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
      601 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
      661 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
      721 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
      781 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
      841 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
      901 gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt
      961 tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac
     1021 taggggttcc ttgtagttaa tgattaaccc gccatgctac ttatctacga tggacattct
     1081 gccattgtga ttactacggt ttggattcct ggggccaagg ggccgtcgtc accgtctcct
     1141 caggtaagaa tggccactct agagcctttg ttttctgcta ttgcctgtgg ggttttctga
     1201 gcattgcagg ctggtcctcg ggacatgttc caagggcacc tgggcggact ggccaggaag
     1261 ggacgggcac tggggtgcct tggggatctg ggagcctctg tggattttcc gatgcctttg
     1321 gaaaatggga cttaggttgg atgtgtctga tggagtaact gagcctgggg gcttggggag
     1381 ccacatttgg acgagatgcc agagcaaacc aggggtctta gtgatggctg aggaatgtgt
     1441 ctcaggtgcg gtgtctgtag gactgcaaga tcgctgcaca cacagcgaat catgaaacat
     1501 tatctttaga attatgagca cactctccgg gacagatata ttccctctaa ccatgatgga
     1561 tattctgaat tacaataaac attgcacgga tgtaggttta tataaattca ctgtgacgaa
     1621 tatatttagc tgctgcccta atgttttgaa tggagatttg acaatttaga taacctggat
     1681 gtttggttgg tttcatataa atcttcaagg gtacaacagc attgaaccta ttccaaaatc
     1741 tatccctgat ccatgatcat actcatctcc cagaccagct ccttcagcac atctccctac
     1801 ctggaagaag aggagtctgg gcttggtgag gggaggcccc aggaagagaa ctgggttctc
     1861 agagggcaca gccagcatcc tcctctcagg gtgagcccca aagactgagg cctccctcat
     1921 cccttttcac ctctccatac aaaggcacca cccacatgca aatcctcact taggcaccca
     1981 caggaaacca ccacacattt ccttaaattc agggtccagc tcacatggga aatgctctct
     2041 gagagtcacg gacctcctgt gcaagaacat ggtgagcaag ggcgaggagc tgttcaccgg
     2101 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
     2161 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
     2221 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
     2281 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     2341 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     2401 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     2461 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     2521 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
     2581 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     2641 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga
     2701 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     2761 tctcggcatg gacgagctgt acaagtaaag cggccgcggg gatccagaca tgataagata
     2821 cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga
     2881 aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa
     2941 caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg tttttttcta
     3001 gagtgcgctg tgtgccaacc tgcatcttaa attctttgtt ggctggaaag agaactgtca
     3061 gagtgggtga atccagccag gacggatgcg tagccttggt cttgatgaga gcagggtcgg
     3121 ggcacgggta gtccagaaag ggtggctgct gtcctgacag aggcttaggg aggccacagg
     3181 agctcagtgc cctgaagttg gtttccaaga gaaaaggatt gttcatctta agaggcgtgc
     3241 ttattaaatg ttaaaagaca ggatatgttt gaagtggctc cagagaaaaa tggttaagac
     3301 aattatgact taaaaatgtg aaagattttg aagtatatta atttttttaa ctgtctaagt
     3361 atttgaaatt cttatcattt gattaacacc catgagtggt atgtctctgg aattagggcc
     3421 aaagtaagtt tagctaagaa atactagcac aggtagataa gtagcatggc gggttaatca
     3481 ttaactacaa ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc
     3541 tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag
     3601 tgagcgagcg agcgcgcagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     3661 aacagttgcg cagcctgaat ggcgaatggc gattccgttg caatggctgg cggtaatatt
     3721 gttctggata ttaccagcaa ggccgatagt ttgagttctt ctactcaggc aagtgatgtt
     3781 attactaatc aaagaagtat tgcgacaacg gttaatttgc gtgatggaca gactctttta
     3841 ctcggtggcc tcactgatta taaaaacact tctcaggatt ctggcgtacc gttcctgtct
     3901 aaaatccctt taatcggcct cctgtttagc tcccgctctg attctaacga ggaaagcacg
     3961 ttatacgtgc tcgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc
     4021 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     4081 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     4141 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     4201 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     4261 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     4321 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     4381 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     4441 aatttaaata tttgcttata caatcttcct gtttttgggg cttttctgat tatcaaccgg
     4501 ggtacatatg attgacatgc tagttttacg attaccgttc atcgattctc ttgtttgctc
     4561 cagactctca ggcaatgacc tgatagcctt tgtagagacc tctcaaaaat agctaccctc
     4621 tccggcatga atttatcagc tagaacggtt aaatatcata ttgatggtga tttgactgtc
     4681 tccggccttt ctcacccgtt tgaatcttta cctacacatt actcaggcat tgcatttaaa
     4741 atatatgagg gttctaaaaa tttttatcct tgcgttgaaa taaaggcttc tcccgcaaaa
     4801 gtattacagg gtcataatgt ttttggtaca accgatttag ctttatgctc tgaggcttta
     4861 ttgcttaatt ttgctaattc tttgccttgc ctgtatgatt tattggatgt tggaatcgcc
     4921 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc
     4981 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     5041 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     5101 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     5161 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     5221 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     5281 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     5341 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     5401 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     5461 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     5521 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     5581 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     5641 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     5701 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     5761 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     5821 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     5881 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     5941 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     6001 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     6061 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     6121 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     6181 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     6241 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     6301 ttgataatc
//
