LOCUS       sequence-102955-        7629 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AdEasy Shuttle vector modified for multicistronic expression. 
            Contains T2A and F2A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7629)
  TITLE     Modified AdEasy shuttle vectors for multicistronic expression
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7629)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pShuttle-CMV-F2A-T2A
FEATURES             Location/Qualifiers
     source          1..7629
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..103
                     /label=ITR
                     /note="inverted terminal repeat of human adenovirus 
                     serotype 5"
     misc_signal     191..341
                     /label=Ad5 Psi
                     /note="packaging signal for adenovirus serotype 5"
     enhancer        399..702
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        703..906
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     857..877
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             969..1034
                     /codon_start=1
                     /product="2A peptide from foot-and-mouth disease virus 
                     polyprotein"
                     /label=F2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="VKQTLNFDLLKLAGDVESNPGP"
     CDS             1065..1118
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     polyA_signal    1277..1398
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1314..1333)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1368..1387
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   4589..4691
                     /label=ITR
                     /note="inverted terminal repeat of human adenovirus 
                     serotype 5"
     primer_bind     complement(4780..4797)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4951..5539)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5031..5050)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             6371..7165
                     /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             6371..7165
                     /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(6425..6444)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7035..7054
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(7213..7592)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7275..7296)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7487..7506
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 catcatcaat aatatacctt attttggatt gaagccaata tgataatgag ggggtggagt
       61 ttgtgacgtg gcgcggggcg tgggaacggg gcgggtgacg tagtagtgtg gcggaagtgt
      121 gatgttgcaa gtgtggcgga acacatgtaa gcgacggatg tggcaaaagt gacgtttttg
      181 gtgtgcgccg gtgtacacag gaagtgacaa ttttcgcgcg gttttaggcg gatgttgtag
      241 taaatttggg cgtaaccgag taagatttgg ccattttcgc gggaaaactg aataagagga
      301 agtgaaatct gaataatttt gtgttactca tagcgcgtaa tactgtaata gtaatcaatt
      361 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
      421 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
      481 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
      541 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
      601 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
      661 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
      721 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
      781 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
      841 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
      901 agagctggtt tagtgaaccg tcagatccgc tagagatctg gtaccgtcga cgcggccgcg
      961 gaagcggagt gaaacagact ttgaattttg accttctcaa gttggcggga gacgtggagt
     1021 ccaaccctgg acctaggcct ggaagcggaa cgcgtggaag cggagagggc agaggaagtc
     1081 tgctaacatg cggtgacgtc gaggagaatc ctggacctct cgagcctaag cttctagata
     1141 agatatccga tccaccggat ctagataact gatcataatc agccatacca catttgtaga
     1201 ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa
     1261 tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     1321 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     1381 actcatcaat gtatcttaac gcggatctgg gcgtggttaa gggtgggaaa gaatatataa
     1441 ggtgggggtc ttatgtagtt ttgtatctgt tttgcagcag ccgccgccgc catgagcacc
     1501 aactcgtttg atggaagcat tgtgagctca tatttgacaa cgcgcatgcc cccatgggcc
     1561 ggggtgcgtc agaatgtgat gggctccagc attgatggtc gccccgtcct gcccgcaaac
     1621 tctactacct tgacctacga gaccgtgtct ggaacgccgt tggagactgc agcctccgcc
     1681 gccgcttcag ccgctgcagc caccgcccgc gggattgtga ctgactttgc tttcctgagc
     1741 ccgcttgcaa gcagtgcagc ttcccgttca tccgcccgcg atgacaagtt gacggctctt
     1801 ttggcacaat tggattcttt gacccgggaa cttaatgtcg tttctcagca gctgttggat
     1861 ctgcgccagc aggtttctgc cctgaaggct tcctcccctc ccaatgcggt ttaaaacata
     1921 aataaaaaac cagactctgt ttggatttgg atcaagcaag tgtcttgctg tctttattta
     1981 ggggttttgc gcgcgcggta ggcccgggac cagcggtctc ggtcgttgag ggtcctgtgt
     2041 attttttcca ggacgtggta aaggtgactc tggatgttca gatacatggg cataagcccg
     2101 tctctggggt ggaggtagca ccactgcaga gcttcatgct gcggggtggt gttgtagatg
     2161 atccagtcgt agcaggagcg ctgggcgtgg tgcctaaaaa tgtctttcag tagcaagctg
     2221 attgccaggg gcaggccctt ggtgtaagtg tttacaaagc ggttaagctg ggatgggtgc
     2281 atacgtgggg atatgagatg catcttggac tgtattttta ggttggctat gttcccagcc
     2341 atatccctcc ggggattcat gttgtgcaga accaccagca cagtgtatcc ggtgcacttg
     2401 ggaaatttgt catgtagctt agaaggaaat gcgtggaaga acttggagac gcccttgtga
     2461 cctccaagat tttccatgca ttcgtccata atgatggcaa tgggcccacg ggcggcggcc
     2521 tgggcgaaga tatttctggg atcactaacg tcatagttgt gttccaggat gagatcgtca
     2581 taggccattt ttacaaagcg cgggcggagg gtgccagact gcggtataat ggttccatcc
     2641 ggcccagggg cgtagttacc ctcacagatt tgcatttccc acgctttgag ttcagatggg
     2701 gggatcatgt ctacctgcgg ggcgatgaag aaaacggttt ccggggtagg ggagatcagc
     2761 tgggaagaaa gcaggttcct gagcagctgc gacttaccgc agccggtggg cccgtaaatc
     2821 acacctatta ccggctgcaa ctggtagtta agagagctgc agctgccgtc atccctgagc
     2881 aggggggcca cttcgttaag catgtccctg actcgcatgt tttccctgac caaatccgcc
     2941 agaaggcgct cgccgcccag cgatagcagt tcttgcaagg aagcaaagtt tttcaacggt
     3001 ttgagaccgt ccgccgtagg catgcttttg agcgtttgac caagcagttc caggcggtcc
     3061 cacagctcgg tcacctgctc tacggcatct cgatccagca tatctcctcg tttcgcgggt
     3121 tggggcggct ttcgctgtac ggcagtagtc ggtgctcgtc cagacgggcc agggtcatgt
     3181 ctttccacgg gcgcagggtc ctcgtcagcg tagtctgggt cacggtgaag gggtgcgctc
     3241 cgggctgcgc gctggccagg gtgcgcttga ggctggtcct gctggtgctg aagcgctgcc
     3301 ggtcttcgcc ctgcgcgtcg gccaggtagc atttgaccat ggtgtcatag tccagcccct
     3361 ccgcggcgtg gcccttggcg cgcagcttgc ccttggagga ggcgccgcac gaggggcagt
     3421 gcagactttt gagggcgtag agcttgggcg cgagaaatac cgattccggg gagtaggcat
     3481 ccgcgccgca ggccccgcag acggtctcgc attccacgag ccaggtgagc tctggccgtt
     3541 cggggtcaaa aaccaggttt cccccatgct ttttgatgcg tttcttacct ctggtttcca
     3601 tgagccggtg tccacgctcg gtgacgaaaa ggctgtccgt gtccccgtat acagacttga
     3661 gagggagttt aaacgaattc aatagcttgt tgcatgggcg gcgatataaa atgcaaggtg
     3721 ctgctcaaaa aatcaggcaa agcctcgcgc aaaaaagaaa gcacatcgta gtcatgctca
     3781 tgcagataaa ggcaggtaag ctccggaacc accacagaaa aagacaccat ttttctctca
     3841 aacatgtctg cgggtttctg cataaacaca aaataaaata acaaaaaaac atttaaacat
     3901 tagaagcctg tcttacaaca ggaaaaacaa cccttataag cataagacgg actacggcca
     3961 tgccggcgtg accgtaaaaa aactggtcac cgtgattaaa aagcaccacc gacagctcct
     4021 cggtcatgtc cggagtcata atgtaagact cggtaaacac atcaggttga ttcacatcgg
     4081 tcagtgctaa aaagcgaccg aaatagcccg ggggaataca tacccgcagg cgtagagaca
     4141 acattacagc ccccatagga ggtataacaa aattaatagg agagaaaaac acataaacac
     4201 ctgaaaaacc ctcctgccta ggcaaaatag caccctcccg ctccagaaca acatacagcg
     4261 cttccacagc ggcagccata acagtcagcc ttaccagtaa aaaagaaaac ctattaaaaa
     4321 aacaccactc gacacggcac cagctcaatc agtcacagtg taaaaaaggg ccaagtgcag
     4381 agcgagtata tataggacta aaaaatgacg taacggttaa agtccacaaa aaacacccag
     4441 aaaaccgcac gcgaacctac gcccagaaac gaaagccaaa aaacccacaa cttcctcaaa
     4501 tcgtcacttc cgttttccca cgttacgtca cttcccattt taagaaaact acaattccca
     4561 acacatacaa gttactccgc cctaaaacct acgtcacccg ccccgttccc acgccccgcg
     4621 ccacgtcaca aactccaccc cctcattatc atattggctt caatccaaaa taaggtatat
     4681 tattgatgat gttaattaac atgcatggat ccatatgcgg tgtgaaatac cgcacagatg
     4741 cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg
     4801 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     4861 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     4921 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     4981 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     5041 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5101 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     5161 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     5221 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     5281 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     5341 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt
     5401 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     5461 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     5521 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     5581 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     5641 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     5701 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     5761 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     5821 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     5881 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     5941 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     6001 tttgcgcaac gttgttgcca ttgctgcagc catgagatta tcaaaaagga tcttcaccta
     6061 gatccttttc acgtagaaag ccagtccgca gaaacggtgc tgaccccgga tgaatgtcag
     6121 ctactgggct atctggacaa gggaaaacgc aagcgcaaag agaaagcagg tagcttgcag
     6181 tgggcttaca tggcgatagc tagactgggc ggttttatgg acagcaagcg aaccggaatt
     6241 gccagctggg gcgccctctg gtaaggttgg gaagccctgc aaagtaaact ggatggcttt
     6301 cttgccgcca aggatctgat ggcgcagggg atcaagctct gatcaagaga caggatgagg
     6361 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     6421 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     6481 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     6541 gaatgaactg caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     6601 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     6661 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     6721 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     6781 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     6841 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag
     6901 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     6961 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     7021 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     7081 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     7141 tcgccttctt gacgagttct tctgaatttt gttaaaattt ttgttaaatc agctcatttt
     7201 ttaaccaata ggccgaaatc ggcaccatcc cttataaatc aaaagaatag accgagatag
     7261 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     7321 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     7381 caagtttttt gtggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     7441 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     7501 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     7561 ccgcgcgctt aatgcgccgc tacagggcgc gtccattcgc cattcaggat cgaattaatt
     7621 cttaattaa
//
