LOCUS       sequence-308674-        5467 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing COM-CD63-4xM12-COM fusion protein in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5467)
  AUTHORS   Yao X, Lyu P, Yoo K, Yadav MK, Singh R, Atala A, Lu B
  TITLE     Engineered extracellular vesicles as versatile ribonucleoprotein 
            delivery vehicles for efficient and safe CRISPR genome editing.
  JOURNAL   J Extracell Vesicles. 2021 Mar;10(5):e12076. doi: 
            10.1002/jev2.12076. Epub 2021 Mar 16.
  PUBMED    33747370
REFERENCE   2  (bases 1 to 5467)
  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: "J Extracell
            Vesicles."; date: "2021-03"; volume: "10(5):e12076. doi"; pages: " 
            10.1002/jev2.12076. Epub 2021 Mar 16"
COMMENT     Sequence Label: pCOM-CD63-4xM12-COM
FEATURES             Location/Qualifiers
     source          1..5467
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        25..228
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     179..199
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     polyA_signal    1799..1920
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1836..1855)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1890..1909
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1927..2382)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2064..2085)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2276..2295
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2409..2513
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2515..2872
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2515..2706
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2538..2558)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2723..2858
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2907..3701
                     /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             2907..3701
                     /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(2961..2980)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3571..3590
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3889..3908)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3933..3980
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4309..4897
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4798..4817
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   4959..5088
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        join(5112..5467,1..24)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
       61 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
      121 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
      181 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg tttagtgaac
      241 cgtcagatcc gctagccacc atgaaatcaa ttcgctgtaa aaactgcaac aaactgttat
      301 ttaaggcgga ttcctttgat cacattgaaa tcaggtgtcc gcgttgcaaa cgtcacatca
      361 taatgctcaa cgcttgtgaa caccctacgg agaaacattg tgggaaaaga gaaaaaatca
      421 cgcattctga cgaaaccgtg cgttatggcg gtcacaactc cggaggcgga ggaggccaat
      481 cccccggccc cgccgctatg gccgtggagg gaggcatgaa gtgcgtgaag tttctgctgt
      541 acgtgctgct gctcgccttc tgtgcttgcg ccgtgggact gattgccgtg ggagtgggag
      601 ctcagctggt gctcagccag acaatcggcg gcggaaggag gcagcctcct agatccatca
      661 gctcccatcc cggcggcggc agaaggcaac ctcctagatc cattagctcc caccccggcg
      721 gaggaaggag acagcccccc agaagcatct cctcccatcc cggcggagga aggagacagc
      781 cccctaggag catttccagc caccccggcg gcggaattca aggcgccaca cccggctctc
      841 tgctgcccgt ggtgattatc gccgtgggtg tcttcctctt cctggtggct tttgtgggct
      901 gctgcggggc ctgcaaggag aactattgtc ttatgatcac gtttgccatc tttctgtctc
      961 ttatcatgtt ggtggaggtg gccgcagcca ttgctggcta tgtgtttaga gataaggtga
     1021 tgtcagagtt taataacaac ttccggcagc agatggagaa ttacccgaaa aataaccaca
     1081 ctgcttcgat cctggacagg atgcaggcag attttaagtg ctgtggggct gctaactaca
     1141 cagattggga gaaaatccct tccatgtcga agaaccgagt ccccgactcc tgctgcatta
     1201 atgttactgt gggctgtggg attaatttca acgagaaggc gatccataag gagggctgtg
     1261 tggagaagat tgggggctgg ctgaggaaaa atgtgctggt ggtagctgca gcagcccttg
     1321 gaattgcttt tgtcgaggtt ttgggaattg tctttgcctg ctgcctcgtg aagagtatca
     1381 gaagtggcta cgaggtgatg tccggtggag gtggatccat ggcttctaac tttactcagt
     1441 tcgttctcgt cgacaatggc ggaactggcg acgtgatgaa atcaattcgc tgtaaaaact
     1501 gcaacaaact gttatttaag gcggattcct ttgatcacat tgaaatcagg tgtccgcgtt
     1561 gcaaacgtca catcataatg ctcaacgctt gtgaacaccc tacggagaaa cattgtggga
     1621 aaagagaaaa aatcacgcat tctgacgaaa ccgtgcgtta ttagagatct gatccaccgg
     1681 atctagataa ctgatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa
     1741 aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa
     1801 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     1861 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     1921 acgcgtaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     1981 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     2041 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     2101 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     2161 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     2221 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     2281 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     2341 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg
     2401 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     2461 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtcctgagg
     2521 cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     2581 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     2641 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     2701 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     2761 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga
     2821 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aagatcgatc
     2881 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     2941 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     3001 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     3061 acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca
     3121 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     3181 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     3241 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     3301 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     3361 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     3421 ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     3481 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     3541 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     3601 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     3661 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga
     3721 aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt
     3781 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     3841 cggggatctc atgctggagt tcttcgccca ccctaggggg aggctaactg aaacacggaa
     3901 ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca
     3961 cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga
     4021 taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca
     4081 ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc
     4141 catagcctca ggttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     4201 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     4261 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     4321 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     4381 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     4441 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     4501 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     4561 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     4621 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     4681 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     4741 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     4801 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     4861 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     4921 acggttcctg gccttttgct ggccttttgc tcacatgtcc tgcaggcagc tgcgcgctcg
     4981 ctcgctcact gaggccgccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg
     5041 agcgagcgcg cagagaggga gtggccaact ccatcactag gggttcctgc ggcctctaga
     5101 ctcgaggcgt tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt
     5161 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
     5221 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
     5281 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
     5341 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
     5401 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
     5461 catctac
//
