LOCUS       Exported                7337 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 7337)
  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..7337
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     LTR             36..180
                     /label=ITR
                     /note="ITR"
     misc_feature    163..180
                     /label=D sequence
     primer_bind     188..200
                     /label=oJW029
     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(442..466)
                     /label=oJW060
     misc_feature    443..462
                     /label=Target sequence
     misc_RNA        463..538
                     /label=gRNA scaffold
     misc_feature    539..549
                     /label=Termination sequence
     primer_bind     625..677
                     /label=AJ19001
     misc_feature    634..653
                     /label=Target sequence
     misc_feature    654..656
                     /label=PAM
     CDS             657..665
                     /codon_start=1
                     /label=Linker
                     /translation="ASG"
     CDS             666..1556
                     /codon_start=1
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIHPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     primer_bind     complement(1544..1591)
                     /label=AJ19002
     CDS             1557..1562
                     /codon_start=1
                     /label=Linker
                     /translation="SA"
     misc_feature    1564..1583
                     /label=Target sequence
     primer_bind     1584..1645
                     /label=KI BB FW1 AJ19019
     misc_feature    1584..1586
                     /label=PAM
     promoter        1630..2808
                     /label=EF-1-alpha promoter
     primer_bind     1705..1724
                     /label=oJWseq5 EF1a FW1
     primer_bind     complement(1968..1984)
                     /label=EF1a rev AJ19025
     primer_bind     2665..2684
                     /label=oJWseq6 EF1a FW2
     primer_bind     complement(2791..2829)
                     /label=BB KI REV1 AJ19020
     primer_bind     2809..2839
                     /label=BB KI FW2 AJ19021
     misc_feature    2809..2818
                     /label=Kozak sequence
     CDS             2821..3525
                     /codon_start=1
                     /label=mCherry
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(2826..2838)
                     /label=pEGFP-RP
                     /note="Macrogen Seq primer"
     primer_bind     complement(3233..3259)
                     /label=BB KI REV2 AJ19022
     primer_bind     3240..3265
                     /label=BB KI FW3
     primer_bind     3463..3481
                     /label=oJWseq8 mCherry-C FW
     CDS             3532..3789
                     /codon_start=1
                     /label=KASH TMD
                     /translation="LRSREEEEETDSRMPHLDSPGSSQPRRSFLSRVIRAALPLQLLLL
                     LLLLLACLLPASEDDYSCTQANNFARSFYPMLRYTNGPPPT"
     primer_bind     3837..3855
                     /label=oJWseq9 hGH polyA FW
     polyA_signal    3842..4317
                     /label=hGH poly(A) signal
     primer_bind     complement(4349..4380)
                     /label=BB KI REV3
     misc_feature    4361..4378
                     /label=D sequence
     LTR             4381..4526
                     /label=ITR
                     /label=ITR(1)
                     /note="ITR"
     rep_origin      complement(4770..5358)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number colE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5529..6389)
                     /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(6390..6494)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6826..7281)
                     /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 cgaatcagag tctctctcgg gcgttttaga gctagaaata
      481 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
      541 ttttgtttta gagctagaaa tagcaagtta aaataaggct agtccgtttt tagcgcgtgc
      601 gccaattctg cagacaaatg gctctagaag cttaatcaga gtctctctcg ggcggggcta
      661 gcggaatggc agaaatcggt actggctttc cattcgaccc ccattatgtg gaagtcctgg
      721 gcgagcgcat gcactacgtc gatgttggtc cgcgcgatgg cacccctgtg ctgttcctgc
      781 acggtaaccc gacctcctcc tacgtgtggc gcaacatcat cccgcatgtt gcaccgaccc
      841 atcgctgcat tgctccagac ctgatcggta tgggcaaatc cgacaaacca gacctgggtt
      901 atttcttcga cgaccacgtc cgcttcatgg atgccttcat cgaagccctg ggtctggaag
      961 aggtcgtcct ggtcattcac gactggggct ccgctctggg tttccactgg gccaagcgca
     1021 atccagagcg cgtcaaaggt attgcattta tggagttcat ccaccctatc ccgacctggg
     1081 acgaatggcc agaatttgcc cgcgagacct tccaggcctt ccgcaccacc gacgtcggcc
     1141 gcaagctgat catcgatcag aacgttttta tcgagggtac gctgccgatg ggtgtcgtcc
     1201 gcccgctgac tgaagtcgag atggaccatt accgcgagcc gttcctgaat cctgttgacc
     1261 gcgagccact gtggcgcttc ccaaacgagc tgccaatcgc cggtgagcca gcgaacatcg
     1321 tcgcgctggt cgaagaatac atggactggc tgcaccagtc ccctgtcccg aagctgctgt
     1381 tctggggcac cccaggcgtt ctgatcccac cggccgaagc cgctcgcctg gccaaaagcc
     1441 tgcctaactg caaggctgtg gacatcggcc cgggtctgaa tctgctgcaa gaagacaacc
     1501 cggacctgat cggcagcgag atcgcgcgct ggctgtccac gctcgagatt tccggcagcg
     1561 ctaaatcaga gtctctctcg ggcgggacgc gccgctagcg gtaccggatc ctacgtatct
     1621 agagtcgacg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag
     1681 aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac
     1741 tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat
     1801 ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag
     1861 gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg
     1921 ccttgaatta cttccacctg gctgcagtac gtgattcttg atcccgagct tcgggttgga
     1981 agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt
     2041 gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt
     2101 ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt
     2161 tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt
     2221 tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg
     2281 ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct
     2341 ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg
     2401 tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca
     2461 aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg
     2521 gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg
     2581 cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt
     2641 tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac
     2701 ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag
     2761 cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgagc caccatgcaa
     2821 gtgagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg
     2881 cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc
     2941 ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc
     3001 gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc
     3061 gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg
     3121 atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc
     3181 gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg
     3241 cagaagaaga ctatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc
     3301 ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag
     3361 gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac
     3421 atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc
     3481 gccgagggcc gccactccac cggcggcatg gacgagctgt acaagtccgg actcagatct
     3541 cgagaggagg aggaggagac agacagcagg atgccccacc tcgacagccc cggcagctcc
     3601 cagccgagac gctccttcct ctcaagggtg atcagggcag cgctaccgtt gcagctgctt
     3661 ctgctgctgc tgctgctcct ggcctgcctg ctacctgcct ctgaagatga ctacagctgc
     3721 acccaggcca acaactttgc ccgatccttc taccccatgc tgcggtacac caacgggcca
     3781 cctcccacct aggaattcga tatcaagctt atcgataccg agcgctgctc gagagatcta
     3841 cgggtggcat ccctgtgacc cctccccagt gcctctcctg gccctggaag ttgccactcc
     3901 agtgcccacc agccttgtcc taataaaatt aagttgcatc attttgtctg actaggtgtc
     3961 cttctataat attatggggt ggaggggggt ggtatggagc aaggggcaag ttgggaagaa
     4021 acctgtaggg cctgcggggt ctattgggaa ccaagctgga gtgcagtggc acaatcttgg
     4081 ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc tgcctcaacc tcccgagttg
     4141 ttgggattcc aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagagacgg
     4201 ggtttcacca tattggccag gctggtctcc aactcctaat ctcaggtgat ctacccacct
     4261 tggcctccca aattgctggg attacaggcg tgaaccactg ctcccttccc tgtccttctg
     4321 attttgtagg taaccacgtg cggaccgagc ggccgatctg aggaacccct agtgatggag
     4381 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgcccgggc aaagcccggg
     4441 cgtcgggcga cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg
     4501 gccaactcca tcactagggg ttccccctgc agcctgcatt aatgaatcgg ccaacgcgcg
     4561 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     4621 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     4681 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     4741 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4801 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4861 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     4921 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg
     4981 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     5041 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     5101 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     5161 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
     5221 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     5281 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     5341 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     5401 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     5461 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     5521 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     5581 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     5641 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     5701 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     5761 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     5821 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     5881 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     5941 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     6001 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     6061 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     6121 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     6181 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     6241 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     6301 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     6361 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     6421 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     6481 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
     6541 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc
     6601 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     6661 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     6721 cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg
     6781 tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggaaatt gtaaacgtta
     6841 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     6901 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     6961 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     7021 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     7081 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     7141 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     7201 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     7261 atgcgccgct acagggcgcg tccattcgcc attcaggctg cgcaactgtt gggaagggcg
     7321 atcggtgcgg gcctctt
//
