LOCUS       Exported                8790 bp ds-DNA     circular SYN 24-FEB-2022
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8790)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Feb 24, 2022 from SnapGene Viewer 4.2.6
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8790
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        162..402
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     162..182
                     /label=hU6 F
     primer_bind     374..403
                     /label=sg1_amp_F
     primer_bind     381..403
                     /label=PCR2_F
     misc_feature    428..520
                     /label=gRNA scaffold1
     primer_bind     complement(428..457)
                     /label=sg1_amp_R
     promoter        546..859
                     /label=mU6
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     656..678
                     /label=mU6 F
     primer_bind     830..859
                     /label=sg2_amp_F
     misc_RNA        899..974
                     /label=gRNA scaffold2
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(899..928)
                     /label=sg2_amp_R
     primer_bind     complement(901..924)
                     /label=PCR2_R
     misc_feature    1030..1147
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1286..2464
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1517..2455
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     CDS             2477..3076
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     misc_feature    3092..3680
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             complement(3563..3574)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3752..3985
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4063..4184
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      4224..4359
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        complement(4380..4398)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4408..4424)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      4566..5021
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        5047..5151
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5152..6012
                     /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      6183..6771
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    7059..7080
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        7095..7125
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7133..7149
                     /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     7157..7173
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        7194..7212
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        7240..7466
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             7467..7647
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    7694..7819
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    8312..8545
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
ORIGIN
        1 taacaaattg gctgtggtat ataaaattat tcataatgat agtaggaggc ttggtaggtt
       61 taagaatagt ttttgctgta ctttctatag tgaatagagt taggcaggga tattcaccat
      121 tatcgtttca gacccacctc ccaaccccga ggggacccag agagggccta tttcccatga
      181 ttccttcata tttgcatata cgatacaagg ctgttagaga gataattaga attaatttga
      241 ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt
      301 agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa
      361 agtatttcga tttcttggct ttatatatct tgtggaaagg actctagaac caggatgggc
      421 accacccgtt taagagctaa gctggaaaca gcatagcaag tttaaataag gctagtccgt
      481 tatcaacttg aaaaagtggc accgagtcgg tgcttttttt gtactgagtc gcccagtctc
      541 agatagatcc gacgccgcca tctctaggcc cgcgccggcc ccctcgcaca gacttgtggg
      601 agaagctcgg ctactcccct gccccggtta atttgcatat aatatttcct agtaactata
      661 gaggcttaat gtgcgataaa agacagataa tctgttcttt ttaatactag ctacatttta
      721 catgataggc ttggatttct ataagagata caaatactaa attattattt taaaaaacag
      781 cacaaaagga aactcaccct aactgtaaag taattgtgtg ttttgagact ataaatatcc
      841 cttggagaaa agccttgttt caccggagac ggcttcttgc tcttttttgt acgtctctgt
      901 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg
      961 caccgagtcg gtgctttttt aagcttggcg taactagatc ttgagacaaa tggcagtatt
     1021 catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt
     1081 agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca
     1141 aaattttcgg gtttattaca gggacagcag agatccactt tggcgccggc tcgagggggc
     1201 ccgggtgcaa agatggataa agttttaaac agagaggaat ctttgcagct aatggacctt
     1261 ctaggtcttg aaaggagtgg gaattggctc cggtgcccgt cagtgggcag agcgcacatc
     1321 gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgatccggtg cctagagaag
     1381 gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg
     1441 tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt
     1501 tgccgccaga acacaggtaa gtgccgtgtg tggttcccgc gggcctggcc tctttacggg
     1561 ttatggccct tgcgtgcctt gaattacttc cacctggctg cagtacgtga ttcttgatcc
     1621 cgagcttcgg gttggaagtg ggtgggagag ttcgaggcct tgcgcttaag gagccccttc
     1681 gcctcgtgct tgagttgagg cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg
     1741 gcaccttcgc gcctgtctcg ctgctttcga taagtctcta gccatttaaa atttttgatg
     1801 acctgctgcg acgctttttt tctggcaaga tagtcttgta aatgcgggcc aagatctgca
     1861 cactggtatt tcggtttttg gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac
     1921 atgttcggcg aggcggggcc tgcgagcgcg gccaccgaga atcggacggg ggtagtctca
     1981 agctggccgg cctgctctgg tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc
     2041 ggcaaggctg gcccggtcgg caccagttgc gtgagcggaa agatggccgc ttcccggccc
     2101 tgctgcaggg agctcaaaat ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc
     2161 cacacaaagg aaaagggcct ttccgtcctc agccgtcgct tcatgtgact ccacggagta
     2221 ccgggcgccg tccaggcacc tcgattagtt ctcgagcttt tggagtacgt cgtctttagg
     2281 ttggggggag gggttttatg cgatggagtt tccccacact gagtgggtgg agactgaagt
     2341 taggccagct tggcacttga tgtaattctc cttggaattt gccctttttg agtttggatc
     2401 ttggttcatt ctcaagcctc agacagtggt tcaaagtttt tttcttccat ttcaggtgtc
     2461 gtgacgtacg gccaccatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga
     2521 cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca
     2581 caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac
     2641 gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg ccgtggcggt
     2701 ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat
     2761 ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc
     2821 gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggagtctcgc ccgaccacca
     2881 gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg
     2941 ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg agcggctcgg
     3001 cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg
     3061 caagcccggt gcctgaacgc gttaagtcga caatcaacct ctggattaca aaatttgtga
     3121 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     3181 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
     3241 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt
     3301 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct
     3361 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg
     3421 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc
     3481 ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg
     3541 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
     3601 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc
     3661 cctttgggcc gcctccccgc gtcgacttta agaccaatga cttacaaggc agctgtagat
     3721 cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga
     3781 caagatctgc tttttgcttg tactgggtct ctctggttag accagatctg agcctgggag
     3841 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     3901 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     3961 tagtcagtgt ggaaaatctc tagcagtacg tatagtagtt catgtcatct tattattcag
     4021 tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt tattgcagct
     4081 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     4141 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggctctag
     4201 ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     4261 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg
     4321 agctattcca gaagtagtga ggaggctttt ttggaggcct agggacgtac ccaattcgcc
     4381 ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga
     4441 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     4501 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     4561 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     4621 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     4681 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     4741 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     4801 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     4861 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     4921 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     4981 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     5041 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     5101 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     5161 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     5221 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     5281 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     5341 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     5401 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     5461 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     5521 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     5581 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     5641 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     5701 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     5761 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     5821 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     5881 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     5941 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     6001 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     6061 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     6121 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     6181 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     6241 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     6301 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     6361 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     6421 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     6481 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     6541 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     6601 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     6661 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     6721 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     6781 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     6841 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     6901 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
     6961 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     7021 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
     7081 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
     7141 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac
     7201 cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct
     7261 tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc
     7321 accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac
     7381 agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat
     7441 ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg
     7501 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc
     7561 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct
     7621 tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg
     7681 gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg
     7741 cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag
     7801 agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat
     7861 tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag
     7921 ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca
     7981 aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata
     8041 taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga
     8101 agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc
     8161 cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata
     8221 aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag
     8281 tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag
     8341 cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat
     8401 tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc
     8461 tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa
     8521 gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca
     8581 ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc
     8641 acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct
     8701 taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata
     8761 aatgggcaag tttgtggaat tggtttaaca
//
