LOCUS       Exported                7494 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 7494)
  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..7494
                     /organism="synthetic DNA construct"
                     /lab_host="Escherichia coli"
                     /mol_type="other DNA"
                     /note="color: #ffffff"
     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"
     promoter        480..720
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
                     /note="color: #ffffff; direction: RIGHT"
     misc_RNA        750..825
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
                     /note="color: #00ccff; direction: RIGHT"
     promoter        839..1079
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
                     /note="color: #ffffff; direction: RIGHT"
     misc_RNA        1109..1184
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
                     /note="color: #00ccff; direction: RIGHT"
     promoter        1198..1438
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
                     /note="color: #ffffff; direction: RIGHT"
     misc_RNA        1468..1543
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
                     /note="color: #00ccff; direction: RIGHT"
     enhancer        1573..1952
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
                     /note="color: #ffffff"
     promoter        1953..2156
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
                     /note="color: #ffffff; direction: RIGHT"
     CDS             2202..2222
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /note="color: #993366"
                     /translation="PKKKRKV"
     misc_feature    2247..4428
                     /label=dCas9N
                     /note="color: #993366; direction: RIGHT"
     misc_feature    4487..4586
                     /label=BDLacZ
                     /note="color: #339966; direction: RIGHT"
     polyA_signal    4633..4714
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
                     /note="color: #a6acb3"
     misc_feature    4801..4931
                     /label=ITR
                     /note="color: #a6acb3"
     primer_bind     complement(4950..4966)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: LEFT"
     rep_origin      5108..5563
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     promoter        5589..5693
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: RIGHT"
     CDS             5694..6554
                     /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: 5694 .. 5762 / #ccffcc / signal sequence
                        2: 5763 .. 6554 / #ccffcc
                     Cleavage site after base 5762"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      6725..7313
                     /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 cgtttaaacg
      481 agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga
      541 taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa
      601 agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata
      661 tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac
      721 gaaacaccga gactccaaga acgccagtcg ttttagagct agaaatagca agttaaaata
      781 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttgtatacga
      841 gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat
      901 aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa
      961 gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat
     1021 gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg
     1081 aaacaccggg gcaccattaa ccactgctgt tttagagcta gaaatagcaa gttaaaataa
     1141 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tgttaacgag
     1201 ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata
     1261 attggaatta atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag
     1321 taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg
     1381 cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga
     1441 aacaccggga agggctccaa gcggaacgtt ttagagctag aaatagcaag ttaaaataag
     1501 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt atttaaatag
     1561 ctagcacgcg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat
     1621 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
     1681 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
     1741 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
     1801 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
     1861 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
     1921 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
     1981 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
     2041 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
     2101 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt
     2161 tagtgaaccg tcagaaccgg tcgactagag gatccatggc cccaaagaag aagcggaagg
     2221 tcggtatcca cggagtccca gcagccgaca agaagtactc cattgggctc gctatcggca
     2281 caaacagcgt cggctgggcc gtcattacgg acgagtacaa ggtgccgagc aaaaaattca
     2341 aagttctggg caataccgat cgccacagca taaagaagaa cctcattggc gccctcctgt
     2401 tcgactccgg ggaaacggcc gaagccacgc ggctcaaaag aacagcacgg cgcagatata
     2461 cccgcagaaa gaatcggatc tgctacctgc aggagatctt tagtaatgag atggctaagg
     2521 tggatgactc tttcttccat aggctggagg agtccttttt ggtggaggag gataaaaagc
     2581 acgagcgcca cccaatcttt ggcaatatcg tggacgaggt ggcgtaccat gaaaagtacc
     2641 caaccatata tcatctgagg aagaagcttg tagacagtac tgataaggct gacttgcggt
     2701 tgatctatct cgcgctggcg catatgatca aatttcgggg acacttcctc atcgaggggg
     2761 acctgaaccc agacaacagc gatgtcgaca aactctttat ccaactggtt cagacttaca
     2821 atcagctttt cgaagagaac ccgatcaacg catccggagt tgacgccaaa gcaatcctga
     2881 gcgctaggct gtccaaatcc cggcggctcg aaaacctcat cgcacagctc cctggggaga
     2941 agaagaacgg cctgtttggt aatcttatcg ccctgtcact cgggctgacc cccaacttta
     3001 aatctaactt cgacctggcc gaagatgcca agcttcaact gagcaaagac acctacgatg
     3061 atgatctcga caatctgctg gcccagatcg gcgaccagta cgcagacctt tttttggcgg
     3121 caaagaacct gtcagacgcc attctgctga gtgatattct gcgagtgaac acggagatca
     3181 ccaaagctcc gctgagcgct agtatgatca agcgctatga tgagcaccac caagacttga
     3241 ctttgctgaa ggcccttgtc agacagcaac tgcctgagaa gtacaaggaa attttcttcg
     3301 atcagtctaa aaatggctac gccggataca ttgacggcgg agcaagccag gaggaatttt
     3361 acaaatttat taagcccatc ttggaaaaaa tggacggcac cgaggagctg ctggtaaagc
     3421 ttaacagaga agatctgttg cgcaaacagc gcactttcga caatggaagc atcccccacc
     3481 agattcacct gggcgaactg cacgctatcc tcaggcggca agaggatttc tacccctttt
     3541 tgaaagataa cagggaaaag attgagaaaa tcctcacatt tcggataccc tactatgtag
     3601 gccccctcgc ccggggaaat tccagattcg cgtggatgac tcgcaaatca gaagagacca
     3661 tcactccctg gaacttcgag gaagtcgtgg ataagggggc ctctgcccag tccttcatcg
     3721 aaaggatgac taactttgat aaaaatctgc ctaacgaaaa ggtgcttcct aaacactctc
     3781 tgctgtacga gtacttcaca gtttataacg agctcaccaa ggtcaaatac gtcacagaag
     3841 ggatgagaaa gccagcattc ctgtctggag agcagaagaa agctatcgtg gacctcctct
     3901 tcaagacgaa ccggaaagtt accgtgaaac agctcaaaga agactatttc aaaaagattg
     3961 aatgtttcga ctctgttgaa atcagcggag tggaggatcg cttcaacgca tccctgggaa
     4021 cgtatcacga tctcctgaaa atcattaaag acaaggactt cctggacaat gaggagaacg
     4081 aggacattct tgaggacatt gtcctcaccc ttacgttgtt tgaagatagg gagatgattg
     4141 aagaacgctt gaaaacttac gctcatctct tcgacgacaa agtcatgaaa cagctcaaga
     4201 ggcgccgata tacaggatgg gggcggctgt caagaaaact gatcaatggg atccgagaca
     4261 agcagagtgg aaagacaatc ctggattttc ttaagtccga tggatttgcc aaccggaact
     4321 tcatgcagtt gatccatgat gactctctca cctttaagga ggacatccag aaagcacaag
     4381 tttctggcca gggggacagt cttcacgagc acatcgctaa tcttgcaggt aagggcactg
     4441 agcagaaggg aagaagctcc gggggctctt tgtagggtgc ggccgcagtg catcaaggcg
     4501 atcacatcag tgaaaaaaag ccagacaggc ggttaaacca acgcagatta aacagcagga
     4561 tgcaaaaatt cgcaggtggt cagatggcgg ccgctctaga ctcgatgagt ttggacaaac
     4621 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
     4681 atttgtaacc attataagct gcaataaaca agttctcgag ttaagggcga attcccgata
     4741 aggatcttcc tagagcatgg ctacgtagat aagtagcatg gcgggttaat cattaactac
     4801 aaggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     4861 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag
     4921 cgagcgcgca gccttaatta acctaattca ctggccgtcg ttttacaacg tcgtgactgg
     4981 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     5041 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     5101 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     5161 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     5221 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     5281 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     5341 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     5401 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     5461 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     5521 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
     5581 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     5641 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     5701 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     5761 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     5821 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     5881 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     5941 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     6001 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     6061 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     6121 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     6181 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     6241 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     6301 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     6361 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     6421 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     6481 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     6541 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     6601 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     6661 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     6721 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     6781 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     6841 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     6901 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     6961 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     7021 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     7081 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     7141 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     7201 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     7261 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     7321 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     7381 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     7441 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaag
//
