pAAVLINK-U6-sgRNA-EF1alpha-5'-ABE8e

LOCUS       pAAVLINK-U6-sgRNA-EF1alpha-5'-ABE8e          6888 bp DNA     circular SYN 10-AUG-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6888)
  AUTHORS   Stavrini Papadaki
  TITLE     Direct Submission
  JOURNAL   Exported Jun 15, 2026 from SnapGene 6.0.2
            https://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..6888
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
                     /note="color: #ffe4c4"
     promoter        145..393
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        145..385
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        415..490
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        504..1046
                     /label=EF1-alpha
                     /note="Chimeric promoter of ef1a and htlv 5' UTR"
     promoter        533..744
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             757..1025
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     misc_feature    851..856
                     /label=SDS
     CDS             1059..4022
                     /codon_start=1
                     /label=ABE8e
                     /translation="MKRTADGSEFESPKKKRKVSEVEFSHEYWMRHALTLAKRARDERE
                     VPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEP
                     CVMCAGAMIHSRIVRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALL
                     CDFYRMPRQVFNAQKKAQSSINSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKKYS
                     IGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLK
                     RTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVD
                     EVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDK
                     LFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLI
                     ALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL
                     LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA
                     GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGEL
                     HAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNF
                     EEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKP
                     AFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHD
                     LLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRR
                     YTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVS
                     GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQK"
     CDS             1095..1115
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1710..3425
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     intron          4023..4063
                     /label=Splicing Donor
     misc_feature    4073..4106
                     /label=lox2272-JT15
     repeat_region   4151..4291
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     misc_feature    4151..4280
                     /label=AAV2 ITR
     repeat_region   4152..4292
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      4366..4821
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        5103..5207
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5208..6068
                     /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"
     rep_origin      6239..6827
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtgagggc ctatttccca tgattccttc atatttgcat
      181 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
      241 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
      301 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
      361 gctttatata tcttgtggaa aggacgaaac accgcccata ccttggagca acgggtttta
      421 gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc
      481 gagtcggtgc tttttttgta caggatctgc gatcgctccg gtgcccgtca gtgggcagag
      541 cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg aacgggtgcc
      601 tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt
      661 cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc
      721 aacgggtttg ccgccagaac acagctgaag cttcgagggg ctcgcatctc tccttcacgc
      781 gcccgccgcc ctacctgagg ccgccatcca cgccggttga gtcgcgttct gccgcctccc
      841 gcctgtggtg cctcctgaac tgcgtccgcc gtctaggtaa gtttaaagct caggtcgaga
      901 ccgggccttt gtccggcgct cccttggagc ctacctagac tcagccggct ctccacgctt
      961 tgcctgaccc tgcttgctca actctacgtc tttgtttcgt tttctgttct gcgccgttac
     1021 agatccaagc tgtgaccggc gcctaccgta cggccaccat gaaacggaca gccgacggaa
     1081 gcgagttcga gtcaccaaag aagaagcgga aagtctctga ggtggagttt tcccacgagt
     1141 actggatgag acatgccctg accctggcca agagggcacg cgatgagagg gaggtgcctg
     1201 tgggagccgt gctggtgctg aacaatagag tgatcggcga gggctggaac agagccatcg
     1261 gcctgcacga cccaacagcc catgccgaaa ttatggccct gagacagggc ggcctggtca
     1321 tgcagaacta cagactgatt gacgccaccc tgtacgtgac attcgagcct tgcgtgatgt
     1381 gcgccggcgc catgatccac tctaggatcg tccgcgtggt gtttggcgtg aggaactcaa
     1441 aaagaggcgc cgcaggctcc ctgatgaacg tgctgaacta ccccggcatg aatcaccgcg
     1501 tcgaaattac cgagggaatc ctggcagatg aatgtgccgc cctgctgtgc gatttctatc
     1561 ggatgcctag acaggtgttc aatgctcaga agaaggccca gagctccatc aactccggag
     1621 gatctagcgg aggctcctct ggctctgaga cacctggcac aagcgagagc gcaacacctg
     1681 aaagcagcgg gggcagcagc ggggggtcag acaagaagta cagcatcggc ctggccatcg
     1741 gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat
     1801 tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc
     1861 tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat
     1921 acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca
     1981 aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga
     2041 agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt
     2101 accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc
     2161 ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg
     2221 gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct
     2281 acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc
     2341 tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg
     2401 agaagaagaa tggcctgttc ggaaacctga ttgccctgag cctgggcctg acccccaact
     2461 tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg
     2521 acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg
     2581 ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga
     2641 tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc
     2701 tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct
     2761 tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt
     2821 tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga
     2881 agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc
     2941 accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat
     3001 tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg
     3061 tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa
     3121 ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca
     3181 tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca
     3241 gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg
     3301 agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc
     3361 tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa
     3421 tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg
     3481 gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa
     3541 acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga
     3601 tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga
     3661 agcggcggag atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg
     3721 acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa
     3781 acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc
     3841 aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg
     3901 ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg
     3961 gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga
     4021 aggtaagtat caaggttaca agacaggttt aaggagacca ataagcgcta gcaattattc
     4081 gtataggata ctttatacga agttattaag ttatggagac cggtgtggca cgtgcggacc
     4141 gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg
     4201 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca
     4261 gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg
     4321 catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg
     4381 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     4441 ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     4501 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     4561 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga
     4621 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     4681 ctggaacaac actcaactct atctcgggct attcttttga tttataaggg attttgccga
     4741 tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     4801 aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat
     4861 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     4921 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     4981 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     5041 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     5101 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     5161 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     5221 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     5281 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     5341 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     5401 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     5461 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     5521 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     5581 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     5641 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     5701 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     5761 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     5821 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     5881 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     5941 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     6001 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     6061 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     6121 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     6181 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     6241 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     6301 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     6361 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     6421 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     6481 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     6541 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     6601 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     6661 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     6721 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     6781 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     6841 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt
//