LOCUS       Exported                7362 bp ds-DNA     circular SYN 10-DEC-2019
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7362)
  AUTHORS   Arthur de Jong
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Jan 29, 2020 from SnapGene Viewer 5.0.6
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7362
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     LTR             36..180
                     /label=ITR
                     /note="ITR"
     misc_feature    163..180
                     /label=D sequence
     promoter        193..433
                     /label=U6 promoter
     primer_bind     193..213
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     364..383
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(374..393)
                     /label=oJWseq4-U6 RV
     misc_feature    413..418
                     /label=TATA box
                     /note="RNA transcription starts 23(+1) bases from the end 
                     of this recognition sequence"
     misc_feature    442..461
                     /label=Target sequence
     misc_RNA        462..537
                     /label=gRNA scaffold
                     /note="in RNA form binds to Cas9"
     misc_feature    538..548
                     /label=Termination sequence
                     /note="RNA polymerase will stop transcription at this 
                     position and fall off"
     misc_feature    634..636
                     /label=PAM
     misc_feature    637..656
                     /label=Target sequence
     CDS             657..689
                     /codon_start=1
                     /label=Linker
                     /translation="GASGSGSSRSG"
     primer_bind     660..702
                     /label=AJ19003
     CDS             690..1580
                     /codon_start=1
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     primer_bind     complement(1569..1616)
                     /label=AJ19004
     misc_feature    1589..1591
                     /label=PAM
     misc_feature    1592..1611
                     /label=Target sequence
     primer_bind     1613..1670
                     /label=KI BB FW1 AJ19019
     promoter        1655..2833
                     /label=EF-1-alpha promoter
     primer_bind     1730..1749
                     /label=oJWseq5 EF1a FW1
     primer_bind     complement(1993..2009)
                     /label=EF1a rev AJ19025
     primer_bind     2690..2709
                     /label=oJWseq6 EF1a FW2
     primer_bind     complement(2816..2854)
                     /label=BB KI REV1 AJ19020
     primer_bind     2834..2864
                     /label=BB KI FW2 AJ19021
     misc_feature    2834..2843
                     /label=Kozak sequence
     CDS             2846..3550
                     /codon_start=1
                     /label=mCherry
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(2851..2863)
                     /label=pEGFP-RP
                     /note="Macrogen Seq primer"
     primer_bind     complement(3258..3284)
                     /label=BB KI REV2 AJ19022
     primer_bind     3265..3290
                     /label=BB KI FW3
     primer_bind     3488..3506
                     /label=oJWseq8 mCherry-C FW
     CDS             3557..3814
                     /codon_start=1
                     /label=KASH TMD
                     /translation="LRSREEEEETDSRMPHLDSPGSSQPRRSFLSRVIRAALPLQLLLL
                     LLLLLACLLPASEDDYSCTQANNFARSFYPMLRYTNGPPPT"
     primer_bind     3862..3880
                     /label=oJWseq9 hGH polyA FW
     polyA_signal    3867..4342
                     /label=hGH poly(A) signal
     primer_bind     complement(4374..4405)
                     /label=BB KI REV3
     misc_feature    4386..4403
                     /label=D sequence
     LTR             4406..4551
                     /label=ITR
                     /label=ITR(1)
                     /note="ITR"
     rep_origin      complement(4795..5383)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number colE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5554..6414)
                     /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"
     promoter        complement(6415..6519)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6851..7306)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 cgctattacg ccaggctgca gggggggggg gggggttggc cactccctct ctgcgcgctc
       61 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg
      121 cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct
      181 cagatcccat gtgagggcct atttcccatg attccttcat atttgcatat acgatacaag
      241 gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat
      301 acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa
      361 atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc
      421 ttgtggaaag gacgaaacac cgggagccac aggattgtaa cgttttagag ctagaaatag
      481 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
      541 tttgttttag agctagaaat agcaagttaa aataaggcta gtccgttttt agcgcgtgcg
      601 ccaattctgc agacaaatgg ctctagaagc ttacccgtta caatcctgtg gctcccggcg
      661 ctagcggtag cggctccagt agatctggga tggcagaaat cggtactggc tttccattcg
      721 acccccatta tgtggaagtc ctgggcgagc gcatgcacta cgtcgatgtt ggtccgcgcg
      781 atggcacccc tgtgctgttc ctgcacggta acccgacctc ctcctacgtg tggcgcaaca
      841 tcatcccgca tgttgcaccg acccatcgct gcattgctcc agacctgatc ggtatgggca
      901 aatccgacaa accagacctg ggttatttct tcgacgacca cgtccgcttc atggatgcct
      961 tcatcgaagc cctgggtctg gaagaggtcg tcctggtcat tcacgactgg ggctccgctc
     1021 tgggtttcca ctgggccaag cgcaatccag agcgcgtcaa aggtattgca tttatggagt
     1081 tcatccgccc tatcccgacc tgggacgaat ggccagaatt tgcccgcgag accttccagg
     1141 ccttccgcac caccgacgtc ggccgcaagc tgatcatcga tcagaacgtt tttatcgagg
     1201 gtacgctgcc gatgggtgtc gtccgcccgc tgactgaagt cgagatggac cattaccgcg
     1261 agccgttcct gaatcctgtt gaccgcgagc cactgtggcg cttcccaaac gagctgccaa
     1321 tcgccggtga gccagcgaac atcgtcgcgc tggtcgaaga atacatggac tggctgcacc
     1381 agtcccctgt cccgaagctg ctgttctggg gcaccccagg cgttctgatc ccaccggccg
     1441 aagccgctcg cctggccaaa agcctgccta actgcaaggc tgtggacatc ggcccgggtc
     1501 tgaatctgct gcaagaagac aacccggacc tgatcggcag cgagatcgcg cgctggctgt
     1561 ccacgctcga gatttccggc taagcgctcc cgttacaatc ctgtggctcc cacgcgccgc
     1621 tagcggtacc ggatcctacg tatctagagt cgacggctcc ggtgcccgtc agtgggcaga
     1681 gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc
     1741 ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt
     1801 tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg
     1861 caacgggttt gccgccagaa cacaggtaag tgccgtgtgt ggttcccgcg ggcctggcct
     1921 ctttacgggt tatggccctt gcgtgccttg aattacttcc acctggctgc agtacgtgat
     1981 tcttgatccc gagcttcggg ttggaagtgg gtgggagagt tcgaggcctt gcgcttaagg
     2041 agccccttcg cctcgtgctt gagttgaggc ctggcctggg cgctggggcc gccgcgtgcg
     2101 aatctggtgg caccttcgcg cctgtctcgc tgctttcgat aagtctctag ccatttaaaa
     2161 tttttgatga cctgctgcga cgcttttttt ctggcaagat agtcttgtaa atgcgggcca
     2221 agatctgcac actggtattt cggtttttgg ggccgcgggc ggcgacgggg cccgtgcgtc
     2281 ccagcgcaca tgttcggcga ggcggggcct gcgagcgcgg ccaccgagaa tcggacgggg
     2341 gtagtctcaa gctggccggc ctgctctggt gcctggcctc gcgccgccgt gtatcgcccc
     2401 gccctgggcg gcaaggctgg cccggtcggc accagttgcg tgagcggaaa gatggccgct
     2461 tcccggccct gctgcaggga gctcaaaatg gaggacgcgg cgctcgggag agcgggcggg
     2521 tgagtcaccc acacaaagga aaagggcctt tccgtcctca gccgtcgctt catgtgactc
     2581 cacggagtac cgggcgccgt ccaggcacct cgattagttc tcgagctttt ggagtacgtc
     2641 gtctttaggt tggggggagg ggttttatgc gatggagttt ccccacactg agtgggtgga
     2701 gactgaagtt aggccagctt ggcacttgat gtaattctcc ttggaatttg ccctttttga
     2761 gtttggatct tggttcattc tcaagcctca gacagtggtt caaagttttt ttcttccatt
     2821 tcaggtgtcg tgagccacca tgcaagtgag caagggcgag gaggataaca tggccatcat
     2881 caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga
     2941 gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt
     3001 gaccaagggt ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg
     3061 ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc
     3121 cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac
     3181 ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa
     3241 cttcccctcc gacggccccg taatgcagaa gaagactatg ggctgggagg cctcctccga
     3301 gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa
     3361 ggacggcggc cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca
     3421 gctgcccggc gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta
     3481 caccatcgtg gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga
     3541 gctgtacaag tccggactca gatctcgaga ggaggaggag gagacagaca gcaggatgcc
     3601 ccacctcgac agccccggca gctcccagcc gagacgctcc ttcctctcaa gggtgatcag
     3661 ggcagcgcta ccgttgcagc tgcttctgct gctgctgctg ctcctggcct gcctgctacc
     3721 tgcctctgaa gatgactaca gctgcaccca ggccaacaac tttgcccgat ccttctaccc
     3781 catgctgcgg tacaccaacg ggccacctcc cacctaggaa ttcgatatca agcttatcga
     3841 taccgagcgc tgctcgagag atctacgggt ggcatccctg tgacccctcc ccagtgcctc
     3901 tcctggccct ggaagttgcc actccagtgc ccaccagcct tgtcctaata aaattaagtt
     3961 gcatcatttt gtctgactag gtgtccttct ataatattat ggggtggagg ggggtggtat
     4021 ggagcaaggg gcaagttggg aagaaacctg tagggcctgc ggggtctatt gggaaccaag
     4081 ctggagtgca gtggcacaat cttggctcac tgcaatctcc gcctcctggg ttcaagcgat
     4141 tctcctgcct caacctcccg agttgttggg attccaggca tgcatgacca ggctcagcta
     4201 atttttgttt ttttggtaga gacggggttt caccatattg gccaggctgg tctccaactc
     4261 ctaatctcag gtgatctacc caccttggcc tcccaaattg ctgggattac aggcgtgaac
     4321 cactgctccc ttccctgtcc ttctgatttt gtaggtaacc acgtgcggac cgagcggccg
     4381 atctgaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     4441 tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt ggtcgcccgg cctcagtgag
     4501 cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttccc cctgcagcct
     4561 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     4621 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     4681 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     4741 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     4801 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     4861 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     4921 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     4981 gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     5041 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     5101 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     5161 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     5221 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     5281 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     5341 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     5401 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     5461 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     5521 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     5581 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     5641 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     5701 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     5761 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     5821 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     5881 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     5941 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     6001 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     6061 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     6121 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     6181 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     6241 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     6301 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     6361 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     6421 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     6481 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     6541 acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac
     6601 gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
     6661 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
     6721 cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat
     6781 tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata
     6841 ccgcatcagg aaattgtaaa cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     6901 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     6961 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     7021 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     7081 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     7141 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     7201 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
     7261 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccat tcgccattca
     7321 ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc tt
//
