A608-Cbh-367Nterm-2A-RT-U6-pegRNA

LOCUS       A608-Cbh-367Nterm-2A-RT-U6-pegRNA            7763 bp DNA     circular SYN 02-APR-2026
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    A608-Cbh-367Nterm-RT-U6-pegRNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7763)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 4, 2026 from SnapGene 6.0.2
            https://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..7763
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
     regulatory      147..950
                     /standard_name="CBh promoter"
                     /label=CBh promoter
                     /note="Geneious type: promoter"
     primer_bind     401..439
                     /label=217-WKG-278-F
     primer_bind     605..625
                     /label=1-CAG-F
     CDS             963..1019
                     /codon_start=1
                     /standard_name="SV40 NLS"
                     /product="nuclear localization signal of SV40 (simianvirus 
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="MKRTADGSEFESPKKKRKV"
     CDS             1020..2117
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N1-367)
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGA"
     primer_bind     1607..1630
                     /label=67-Cas9-F1
     primer_bind     complement(1607..1630)
                     /label=116-WKG-338-R3
     primer_bind     complement(1736..1752)
                     /label=78-Cas9-R1
     primer_bind     complement(2088..2110)
                     /label=115-WKG-338-R2
     CDS             2118..2423
                     /codon_start=1
                     /label=RmaDnaBIntN
                     /translation="CLAGDTLITLADGRRVPIRELVSQQNFSVWALNPQTYRLERARVS
                     RAFCTGIKPVYRLTTRLGRSIRATANHRFLTPQGWKRVDELQPGDYLALPRRIPTAS"
     CDS             2424..2486
                     /codon_start=1
                     /label=NLS
                     /label=BPNLS
                     /note="/Transferred_From=C-terminal BPNLS"
                     /note="/Transferred_Similarity=100.00%"
                     /note="/created_by=jonlevy"
                     /note="/modified_by=jonlevy"
                     /translation="SGGSKRTADGSEFEPKKKRKV"
     CDS             2487..2543
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2544..2621
                     /codon_start=1
                     /label=SV40 NLS-2xSGGS
                     /translation="KRTADGSEFESPKKKRKVSGGSSGGS"
     CDS             2622..4112
                     /standard_name="RT codon optimized Genscript"
                     /label=RT codon optimized Genscript
     CDS             4125..4178
                     /standard_name="SV40 NLS"
                     /product="nuclear localization signal of SV40 (simianvirus 
                     40) large T antigen"
                     /label=SV40 NLS
     misc_feature    4188..4311
                     /label=SV40 polyA
     ncRNA           complement(4373..4409)
                     /standard_name="evoPQ1-trimmed"
                     /label=evoPQ1-trimmed
     misc_feature    complement(4445..4530)
                     /label=RNA scaffold
     regulatory      complement(4552..4815)
                     /standard_name="hU6 promoter"
                     /label=hU6 promoter
                     /note="Geneious type: promoter"
     primer_bind     4778..4795
                     /label=481-hU6-F
     repeat_region   4824..4953
                     /label=AAV2 ITR
     primer_bind     complement(4977..4995)
                     /label=43-WKG254-F
     rep_origin      5130..5585
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5144..5168)
                     /label=184-WKG-599-R
     primer_bind     complement(5187..5206)
                     /label=121-WKG-450-F1
     primer_bind     complement(5219..5239)
                     /label=122-WKG-465-F
     primer_bind     5429..5448
                     /label=189-WKG-612-F
     promoter        5611..5715
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5716..6576
                     /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(6208..6230)
                     /label=198-WKG-623-F4
     rep_origin      6747..7335
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6823..6851)
                     /label=197-WKG-623-F3
     primer_bind     7224..7248
                     /label=225-WKG-1079-F
     primer_bind     7350..7369
                     /label=422-PM1195-F1
     primer_bind     7414..7440
                     /label=79-WKG-304-F1
     primer_bind     complement(7442..7461)
                     /label=196-WKG-623-F2
     primer_bind     7555..7589
                     /label=139-WKG-445-R12
     protein_bind    7623..7644
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     7646..7668
                     /label=154-lacUV5 prom-For
     primer_bind     7653..7676
                     /label=140-WKG-448-R1
     primer_bind     7653..7676
                     /label=190-WKG-612-R
     primer_bind     7656..7678
                     /label=41-lac-R
     promoter        7659..7689
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7697..7713
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     7726..7741
                     /label=152-M13-Rev
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcctcta gatcagggta cccgttacat aacttacggt aaatggcccg
      181 cctggctgac cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc aatagggact
      241 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
      301 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
      361 cattgtgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
      421 gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc
      481 cccccctccc cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg
      541 ggggcggggg gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc
      601 ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct
      661 tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga
      721 gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc
      781 cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg
      841 ggctgtaatt agctgagcaa gaggtaaggg tttaagggat ggttggttgg tggggtatta
      901 atgtttaatt acctggagca cctgcctgaa atcacttttt ttcaggttgg accggtgcca
      961 ccatgaaacg gacagccgac ggaagcgagt tcgagtcacc aaagaagaag cggaaagtcg
     1021 acaagaagta cagcatcggc ctggacatcg gcaccaactc tgtgggctgg gccgtgatca
     1081 ccgacgagta caaggtgccc agcaagaaat tcaaggtgct gggcaacacc gaccggcaca
     1141 gcatcaagaa gaacctgatc ggagccctgc tgttcgacag cggcgaaaca gccgaggcca
     1201 cccggctgaa gagaaccgcc agaagaagat acaccagacg gaagaaccgg atctgctatc
     1261 tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc cacagactgg
     1321 aagagtcctt cctggtggaa gaggataaga agcacgagcg gcaccccatc ttcggcaaca
     1381 tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac
     1441 tggtggacag caccgacaag gccgacctgc ggctgatcta tctggccctg gcccacatga
     1501 tcaagttccg gggccacttc ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg
     1561 acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa aaccccatca
     1621 acgccagcgg cgtggacgcc aaggccatcc tgtctgccag actgagcaag agcagacggc
     1681 tggaaaatct gatcgcccag ctgcccggcg agaagaagaa tggcctgttc ggaaacctga
     1741 ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg gccgaggatg
     1801 ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg ctggcccaga
     1861 tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc
     1921 tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc gcctctatga
     1981 tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc
     2041 agctgcctga gaagtacaaa gagattttct tcgaccagag caagaacggc tacgccggct
     2101 acattgacgg cggagcctgt ctggctggcg atactctcat taccctggcc gatggacgac
     2161 gagtgcctat tagagaactg gtgtcacagc agaatttttc cgtgtgggct ctgaatcctc
     2221 agacttaccg cctggagagg gctagagtga gtagagcttt ctgtaccggc atcaaacctg
     2281 tgtaccgcct caccactaga ctggggagat ccattagggc cactgccaac caccgatttc
     2341 tcacacctca gggctggaaa cgagtcgatg aactccagcc tggagattac ctggctctgc
     2401 ctaggagaat ccctactgcc tccagcggag gcagtaaaag aacagcagac gggagtgagt
     2461 ttgagcccaa gaaaaagaga aaggtggcta ccaacttcag cctcctgaag caggctggcg
     2521 acgtggagga aaatcctggc ccaaagcgga ccgccgacgg cagcgaattc gagagcccta
     2581 agaagaaaag aaaggtgagc ggaggctcta gcggcggaag caccctgaac attgaagacg
     2641 agtatagact gcatgaaaca agcaaggaac ccgacgtgtc cctgggctcc acctggctgt
     2701 ccgactttcc ccaggcctgg gccgagacag gaggaatggg cctggccgtg cggcaggcac
     2761 ccctgatcat ccctctgaag gccacctcta cacccgtgag catcaagcag taccctatgt
     2821 ctcaggaggc cagactgggc atcaagcctc acatccagag gctgctggac cagggcatcc
     2881 tggtgccatg ccagagcccc tggaacacac cactgctgcc cgtgaagaag ccaggcacca
     2941 atgactatag acccgtgcag gatctgagag aggtgaacaa gagggtggag gatatccacc
     3001 ccaccgtgcc caacccttac aatctgctgt ccggcctgcc cccttctcac cagtggtata
     3061 cagtgctgga cctgaaggat gccttctttt gtctgagact gcaccctacc agccagccac
     3121 tgttcgcctt tgagtggagg gaccctgaga tgggcatctc tggccagctg acctggacac
     3181 gcctgcctca gggcttcaag aatagcccaa cactgtttaa cgaggccctg caccgcgacc
     3241 tggcagattt ccggatccag cacccagatc tgatcctgct gcagtacgtg gacgatctgc
     3301 tgctggccgc caccagcgag ctggattgcc agcagggaac acgcgccctg ctgcagaccc
     3361 tgggaaacct gggatatagg gcatccgcca agaaggccca gatctgtcag aagcaggtga
     3421 agtacctggg ctatctgctg aaggagggcc agagatggct gacagaggcc aggaaggaga
     3481 cagtgatggg ccagccaaca cccaagaccc caagacagct gagggagttc ctgggcaaag
     3541 caggattttg caggctgttc atcccaggat tcgcagagat ggcagcacct ctgtacccac
     3601 tgaccaagcc gggcaccctg tttaattggg gccctgacca gcagaaggcc tatcaggaga
     3661 tcaagcaggc cctgctgaca gcaccagccc tgggcctgcc agacctgacc aagcctttcg
     3721 agctgtttgt ggatgagaag cagggctacg ccaagggcgt gctgacccag aagctgggac
     3781 catggagacg gcccgtggcc tatctgtcca agaagctgga cccagtggca gcaggatggc
     3841 caccatgcct gaggatggtg gcagcaatcg ccgtgctgac aaaggatgcc ggcaagctga
     3901 ccatgggaca gccactggtc atcctggcac cacacgcagt ggaggccctg gtgaagcagc
     3961 ctccagatcg ctggctgtct aacgcccgga tgacacacta ccaggccctg ctgctggaca
     4021 ccgatcgcgt gcagtttggc cctgtggtgg ccctgaatcc agccaccctg ctgcctctgc
     4081 cagaggaggg cctgcagcac aactgtctgg actctggcgg ctcaaaaaga accgccgacg
     4141 gcagcgaatt cgagtctccc aagaagaaga ggaaagtcta agtcgacctt tatttgtgaa
     4201 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac
     4261 aacaattgca ttcattttat gtttcaggtt cagggggaga tgtgggaggt tcacaaataa
     4321 agcatttttt tcactgcggg gatgcggtgg gctctatggc tcgagaaaaa aattctagtt
     4381 ggtttaacgc gtaactagat agaaccgcgt atttaaacca tgggatggag tgggcgaaga
     4441 caaaggaccg actcggtccc actttttcaa gttgataacg gactagcctt atttaaactt
     4501 gctatgctgt ttccagcata gctcttaaac gagcatccca tggaacctgg cggtgtttcg
     4561 tcctttccac aagatatata aagccaagaa atcgaaatac tttcaagtta cggtaagcat
     4621 atgatagtcc attttaaaac ataattttaa aactgcaaac tacccaagaa attattactt
     4681 tctacgtcac gtattttgta ctaatatctt tgtgtttaca gtcaaattaa ttctaattat
     4741 ctctctaaca gccttgtatc gtatatgcaa atatgaagga atcatgggaa ataggccctc
     4801 ttcctgcccg accttgcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct
     4861 gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc
     4921 ccgggcggcc tcagtgagcg agcgagcgcg cagccttaat taacctaatt cactggccgt
     4981 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc
     5041 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca
     5101 acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
     5161 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     5221 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     5281 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     5341 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     5401 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     5461 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     5521 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     5581 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     5641 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     5701 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     5761 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     5821 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     5881 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     5941 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     6001 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     6061 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     6121 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     6181 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     6241 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     6301 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     6361 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     6421 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     6481 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     6541 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     6601 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     6661 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     6721 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     6781 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     6841 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     6901 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     6961 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     7021 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     7081 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     7141 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     7201 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     7261 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     7321 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     7381 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     7441 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     7501 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     7561 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     7621 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
     7681 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat
     7741 gattacgcca gatttaatta agg
//