LOCUS       Exported                7709 bp DNA     circular SYN 14-FEB-2024
DEFINITION  Sequenced and manually curated.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7709)
  AUTHORS   Balint Szalontai
  TITLE     Direct Submission
  JOURNAL   Exported Feb 14, 2024 from SnapGene 5.3.3
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7709
                     /organism="synthetic DNA construct"
                     /lab_host="Escherichia coli"
                     /mol_type="other DNA"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
                     /note="color: #31849b"
     promoter        143..173
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
                     /note="This forward directional feature has 3 segments:
                        1: 143 .. 148 / #ffffff / -35
                        2: 149 .. 166 / #ffffff
                        3: 167 .. 173 / #ffffff / -10"
     protein_bind    181..197
                     /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)."
                     /note="color: #31849b"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: RIGHT"
     misc_feature    248..389
                     /label=ITR
                     /note="color: #a6acb3"
     enhancer        494..873
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
                     /note="color: #ffffff"
     promoter        874..1077
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
                     /note="color: #ffffff; direction: RIGHT"
     misc_feature    complement(1135..1234)
                     /label=BDLacZ
                     /note="color: #339966; direction: LEFT"
     misc_feature    1235..3218
                     /label=dCas9C(m4)
                     /note="color: #993366"
     misc_feature    1243..1266
                     /label=ASS620
                     /note="color: #800080"
     primer_bind     1478..1497
                     /label=Cas9 mut seq fw
                     /note="color: black; sequence: CCCAGTTGAAAACACCCAGC; added:
                     2021-01-19"
     misc_feature    1599..1600
                     /label=GC
                     /note="color: #a6acb3"
     primer_bind     2161..2180
                     /label=qPCR 3' Cas9 fw
                     /note="color: black; sequence: AGGAAATAGGCAAGGCCACC; added:
                     2021-03-10"
     primer_bind     complement(2253..2272)
                     /label=qPCR 3' Cas9 rev
                     /note="color: black; sequence: TCGATAAGTGGTCGCTTCCG; added:
                     2021-03-10"
     CDS             3198..3218
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /note="color: #993366"
                     /translation="PKKKRKV"
     protein_bind    3255..3279
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
                     /note="color: #31849b; direction: RIGHT"
     CDS             3309..3458
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /note="color: #993366"
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             3483..3503
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /note="color: #993366"
                     /translation="PKKKRKV"
     CDS             3933..4289
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /note="color: #993366"
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALL"
     CDS             4308..4877
                     /codon_start=1
                     /product="transcriptional activation domain from the human 
                     herpesvirus 4 (Epstein-Barr virus) replication and 
                     transcription activator Rta/BRLF1 (Hardwick et al., 1992; 
                     Chavez et al., 2015)"
                     /label=Rta AD
                     /note="color: #993366"
                     /translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
                     RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
                     REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
                     DTFLNDECLLHAMHISTGLSIFDTSLF"
     polyA_signal    4882..4929
                     /label=synth polyA signal
                     /note="mRNA trans-splicing dual AAV vectors for (epi)genome
                     editing and gene therapy | Nature Communications"
                     /note="color: #a6acb3; direction: RIGHT"
     primer_bind     complement(4929..4951)
                     /label=Primer 1
                     /note="color: black; sequence: Gggaattcgcccttaactcgagaac; 
                     added: 2024-02-06"
     misc_feature    5016..5146
                     /label=ITR
                     /note="color: #a6acb3"
     primer_bind     complement(5165..5181)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: LEFT"
     rep_origin      5323..5778
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     promoter        5804..5908
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: RIGHT"
     CDS             5909..6769
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="This feature has 2 segments:
                        1: 5909 .. 5977 / #ccffcc / signal sequence
                        2: 5978 .. 6769 / #ccffcc
                     Cleavage site after base 5977"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      6940..7528
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccagattta
      241 attaaggcct taattaggct gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc
      301 gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga
      361 gtggccaact ccatcactag gggttccttg tagttaatga ttaacccgcc atgctactta
      421 tctacgtagc catgctctag gaagatcgga attcgccctt aagggcgcgc cgtttaaata
      481 gctagcacgc gttgacattg attattgact agttattaat agtaatcaat tacggggtca
      541 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      601 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      661 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      721 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      781 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      841 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      901 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      961 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
     1021 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt
     1081 ttagtgaacc gtcagaggta ccaccggtcg actagaggat ccaggtgcgg ccgccatctg
     1141 accacctgcg aatttttgca tcctgctgtt taatctgcgt tggtttaacc gcctgtctgg
     1201 ctttttttca ctgatgtgat cgccttgatg cactgcggcc gccaacgagt cttttgtcat
     1261 ctacaggtag cccagctatc aaaaagggaa tactgcagac cgttaaggtc gtggatgaac
     1321 tcgtcaaagt aatgggaagg cataagcccg agaatatcgt tatcgagatg gcccgagaga
     1381 accaaactac ccagaaggga cagaagaaca gtagggaaag gatgaagagg attgaagagg
     1441 gtataaaaga actggggtcc caaatcctta aggaacaccc agttgaaaac acccagcttc
     1501 agaatgagaa gctctacctg tactacctgc agaacggcag ggacatgtac gtggatcagg
     1561 aactggacat caatcggctc tccgactacg acgtggctgc tatcgtgccc cagtcttttc
     1621 tcaaagatga ttctattgat aataaagtgt tgacaagatc cgataaagct agagggaaga
     1681 gtgataacgt cccctcagaa gaagttgtca agaaaatgaa aaattattgg cggcagctgc
     1741 tgaacgccaa actgatcaca caacggaagt tcgataatct gactaaggct gaacgaggtg
     1801 gcctgtctga gttggataaa gccggcttca tcaaaaggca gcttgttgag acacgccaga
     1861 tcaccaagca cgtggcccaa attctcgatt cacgcatgaa caccaagtac gatgaaaatg
     1921 acaaactgat tcgagaggtg aaagttatta ctctgaagtc taagctggtc tcagatttca
     1981 gaaaggactt tcagttttat aaggtgagag agatcaacaa ttaccaccat gcgcatgatg
     2041 cctacctgaa tgcagtggta ggcactgcac ttatcaaaaa atatcccaag cttgaatctg
     2101 aatttgttta cggagactat aaagtgtacg atgttaggaa aatgatcgca aagtctgagc
     2161 aggaaatagg caaggccacc gctaagtact tcttttacag caatattatg aattttttca
     2221 agaccgagat tacactggcc aatggagaga ttcggaagcg accacttatc gaaacaaacg
     2281 gagaaacagg agaaatcgtg tgggacaagg gtagggattt cgcgacagtc cggaaggtcc
     2341 tgtccatgcc gcaggtgaac atcgttaaaa agaccgaagt acagaccgga ggcttctcca
     2401 aggaaagtat cctcccgaaa aggaacagcg acaagctgat cgcacgcaaa aaagattggg
     2461 accccaagaa atacggcgga ttcgattctc ctacagtcgc ttacagtgta ctggttgtgg
     2521 ccaaagtgga gaaagggaag tctaaaaaac tcaaaagcgt caaggaactg ctgggcatca
     2581 caatcatgga gcgatcaagc ttcgaaaaaa accccatcga ctttctcgag gcgaaaggat
     2641 ataaagaggt caaaaaagac ctcatcatta agcttcccaa gtactctctc tttgagcttg
     2701 aaaacggccg gaaacgaatg ctcgctagtg cgggcgagct gcagaaaggt aacgagctgg
     2761 cactgccctc taaatacgtt aatttcttgt atctggccag ccactatgaa aagctcaaag
     2821 ggtctcccga agataatgag cagaagcagc tgttcgtgga acaacacaaa cactaccttg
     2881 atgagatcat cgagcaaata agcgaattct ccaaaagagt gatcctcgcc gacgctaacc
     2941 tcgataaggt gctttctgct tacaataagc acagggataa gcccatcagg gagcaggcag
     3001 aaaacattat ccacttgttt actctgacca acttgggcgc gcctgcagcc ttcaagtact
     3061 tcgacaccac catagacaga aagcggtaca cctctacaaa ggaggtcctg gacgccacac
     3121 tgattcatca gtcaattacg gggctctatg aaacaagaat cgacctctct cagctcggtg
     3181 gagacagcag ggctgacccc aagaagaaga ggaaggtgtc gccagggatc cgtcgacttg
     3241 acgcgttgat atcaacaagt ttgtacaaaa aagcaggcta caaagaggcc agcggttccg
     3301 gacgggctga cgcattggac gattttgatc tggatatgct gggaagtgac gccctcgatg
     3361 attttgacct tgacatgctt ggttcggatg cccttgatga ctttgacctc gacatgctcg
     3421 gcagtgacgc ccttgatgat ttcgacctgg acatgctgat taactctaga agttccggat
     3481 ctccgaaaaa gaaacgcaaa gttggtagcc agtacctgcc cgacaccgac gaccggcacc
     3541 ggatcgagga aaagcggaag cggacctacg agacattcaa gagcatcatg aagaagtccc
     3601 ccttcagcgg ccccaccgac cctagacctc cacctagaag aatcgccgtg cccagcagat
     3661 ccagcgccag cgtgccaaaa cctgcccccc agccttaccc cttcaccagc agcctgagca
     3721 ccatcaacta cgacgagttc cctaccatgg tgttccccag cggccagatc tctcaggcct
     3781 ctgctctggc tccagcccct cctcaggtgc tgcctcaggc tcctgctcct gcaccagctc
     3841 cagccatggt gtctgcactg gctcaggcac cagcacccgt gcctgtgctg gctcctggac
     3901 ctccacaggc tgtggctcca ccagccccta aacctacaca ggccggcgag ggcacactgt
     3961 ctgaagctct gctgcagctg cagttcgacg acgaggatct gggagccctg ctgggaaaca
     4021 gcaccgatcc tgccgtgttc accgacctgg ccagcgtgga caacagcgag ttccagcagc
     4081 tgctgaacca gggcatccct gtggcccctc acaccaccga gcccatgctg atggaatacc
     4141 ccgaggccat cacccggctc gtgacaggcg ctcagaggcc tcctgatcca gctcctgccc
     4201 ctctgggagc accaggcctg cctaatggac tgctgtctgg cgacgaggac ttcagctcta
     4261 tcgccgatat ggatttctca gccttgctgg gctctggcag cggcagccgg gattccaggg
     4321 aagggatgtt tttgccgaag cctgaggccg gctccgctat tagtgacgtg tttgagggcc
     4381 gcgaggtgtg ccagccaaaa cgaatccggc catttcatcc tccaggaagt ccatgggcca
     4441 accgcccact ccccgccagc ctcgcaccaa caccaaccgg tccagtacat gagccagtcg
     4501 ggtcactgac cccggcacca gtccctcagc cactggatcc agcgcccgca gtgactcccg
     4561 aggccagtca cctgttggag gatcccgatg aagagacgag ccaggctgtc aaagcccttc
     4621 gggagatggc cgatactgtg attccccaga aggaagaggc tgcaatctgt ggccaaatgg
     4681 acctttccca tccgccccca aggggccatc tggatgagct gacaaccaca cttgagtcca
     4741 tgaccgagga tctgaacctg gactcacccc tgaccccgga attgaacgag attctggata
     4801 ccttcctgaa cgacgagtgc ctcttgcatg ccatgcatat cagcacagga ctgtccatct
     4861 tcgacacatc tctgttttga caataaaata tctttatttt cattacatct gtgtgttggt
     4921 tttttgtgtc tcgagttaag ggcgaattcc cgataaggat cttcctagag catggctacg
     4981 tagataagta gcatggcggg ttaatcatta actacaagga acccctagtg atggagttgg
     5041 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac
     5101 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagcctt aattaaccta
     5161 attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     5221 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
     5281 atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg
     5341 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     5401 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     5461 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     5521 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     5581 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     5641 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     5701 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     5761 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     5821 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     5881 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     5941 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     6001 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     6061 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     6121 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     6181 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     6241 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     6301 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     6361 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     6421 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     6481 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     6541 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     6601 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     6661 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     6721 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     6781 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     6841 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     6901 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     6961 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     7021 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     7081 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     7141 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     7201 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     7261 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     7321 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     7381 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     7441 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     7501 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     7561 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     7621 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     7681 cagcgagtca gtgagcgagg aagcggaag
//
