LOCUS       sequence-479509-        6175 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For targeted gene knock-in at NHP IGH locus and constitutive 
            expression of GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6175)
  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 6175)
  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: pCMVp_eGFP
FEATURES             Location/Qualifiers
     source          1..6175
                     /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
     promoter        1691..1888
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1845..1865
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(1935..1937,1938..1940,1941..2654)
                     /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(1935..1937,1938..1940,1941..2651)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1980..2001)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2241..2260)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2588..2609
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2667..2801
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2691..2710)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2745..2764
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3356..3485
                     /label=AAV2 ITR
     rep_origin      3856..4369
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3943..3962)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4153..4174
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4851..4870)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4970..4992
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5030..5048)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5116..5220
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5221..5289,5290..6081)
                     /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(5439..5458)
                     /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 attatgagcg ttacataact tacggtaaga ggcccgcctg gctgaccgcc
     1561 caacgacccc cgcccattga cgtcaataat gacgtatgtt tccatagtaa cgccaatagg
     1621 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tagtcatcgc
     1681 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
     1741 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
     1801 ccaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
     1861 gcgtgtacgg gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
     1921 ccggtcgcaa gaacatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     1981 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
     2041 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
     2101 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
     2161 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     2221 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     2281 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     2341 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     2401 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     2461 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     2521 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
     2581 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     2641 agctgtacaa gtaaagcggc cgcggggatc cagacatgat aagatacatt gatgagtttg
     2701 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
     2761 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
     2821 attttatgtt tcaggttcag ggggaggtgt gggaggtttt tttctagagt gcgctgtgtg
     2881 ccaacctgca tcttaaattc tttgttggct ggaaagagaa ctgtcagagt gggtgaatcc
     2941 agccaggacg gatgcgtagc cttggtcttg atgagagcag ggtcggggca cgggtagtcc
     3001 agaaagggtg gctgctgtcc tgacagaggc ttagggaggc cacaggagct cagtgccctg
     3061 aagttggttt ccaagagaaa aggattgttc atcttaagag gcgtgcttat taaatgttaa
     3121 aagacaggat atgtttgaag tggctccaga gaaaaatggt taagacaatt atgacttaaa
     3181 aatgtgaaag attttgaagt atattaattt ttttaactgt ctaagtattt gaaattctta
     3241 tcatttgatt aacacccatg agtggtatgt ctctggaatt agggccaaag taagtttagc
     3301 taagaaatac tagcacaggt agataagtag catggcgggt taatcattaa ctacaaggaa
     3361 cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg
     3421 cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg
     3481 cgcagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     3541 ctgaatggcg aatggcgatt ccgttgcaat ggctggcggt aatattgttc tggatattac
     3601 cagcaaggcc gatagtttga gttcttctac tcaggcaagt gatgttatta ctaatcaaag
     3661 aagtattgcg acaacggtta atttgcgtga tggacagact cttttactcg gtggcctcac
     3721 tgattataaa aacacttctc aggattctgg cgtaccgttc ctgtctaaaa tccctttaat
     3781 cggcctcctg tttagctccc gctctgattc taacgaggaa agcacgttat acgtgctcgt
     3841 caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     3901 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     3961 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     4021 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     4081 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     4141 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     4201 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     4261 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taaatatttg
     4321 cttatacaat cttcctgttt ttggggcttt tctgattatc aaccggggta catatgattg
     4381 acatgctagt tttacgatta ccgttcatcg attctcttgt ttgctccaga ctctcaggca
     4441 atgacctgat agcctttgta gagacctctc aaaaatagct accctctccg gcatgaattt
     4501 atcagctaga acggttaaat atcatattga tggtgatttg actgtctccg gcctttctca
     4561 cccgtttgaa tctttaccta cacattactc aggcattgca tttaaaatat atgagggttc
     4621 taaaaatttt tatccttgcg ttgaaataaa ggcttctccc gcaaaagtat tacagggtca
     4681 taatgttttt ggtacaaccg atttagcttt atgctctgag gctttattgc ttaattttgc
     4741 taattctttg ccttgcctgt atgatttatt ggatgttgga atcgcctgat gcggtatttt
     4801 ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc
     4861 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
     4921 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     4981 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata
     5041 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     5101 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     5161 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     5221 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     5281 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     5341 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     5401 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     5461 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     5521 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     5581 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     5641 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     5701 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     5761 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     5821 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     5881 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     5941 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     6001 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     6061 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     6121 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatc
//
