LOCUS       Exported                4483 bp DNA     circular SYN 22-OCT-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4483)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Mar 1, 2022 from SnapGene 5.3.2
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4483
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     27..86
                     /label=Cre-OFF FW
     protein_bind    39..72
                     /label=loxP511
     primer_bind     72..85
                     /label=FW
     misc_feature    73..381
                     /label=mU6*
                     /note="see Tiscornia PNAS 
                     2004https://www.pnas.org/content/101/19/7347
                     "
     primer_bind     complement(330..381)
                     /label=Cre-OFF REV
     protein_bind    343..376
                     /label=loxP511
     misc_feature    357..362
                     /label=TATA
     misc_feature    363..385
                     /label=Tata + 23
     primer_bind     369..424
                     /label=Cre-OFF Fill in
     primer_bind     382..405
                     /label=WD0096 Homer1 i17 g8757 gRNA FW
     misc_feature    386..405
                     /label=target sequence
     misc_RNA        406..481
                     /label=gRNA scaffold
     misc_feature    482..492
                     /label=Termination sequence
     primer_bind     487..506
                     /label=Cre-OFF Fill in
     misc_feature    566..571
                     /label=Multiple cloning site
     primer_bind     571..616
                     /label=WD0116 Homer1 e8 g8757 FW
     misc_feature    572..575
                     /label=HindIII
     misc_feature    573..1695
                     /label=sequenced 14-4-2021
     primer_bind     575..595
                     /label=WD0096 Homer1 i17 g8757 gRNA FW
     misc_feature    576..595
                     /label=targeting sequence
     misc_feature    596..598
                     /label=PAM
     misc_feature    599..708
                     /label=CRISPIE acceptor (upstream of insert)
     misc_feature    599..696
                     /label=junctional intron
     misc_feature    697..698
                     /label=splice acceptor
     misc_feature    699..708
                     /label=junctional exon
     CDS             709..717
                     /codon_start=1
                     /label=Linker w/ NheI
                     /translation="ASG"
     CDS             718..1608
                     /codon_start=1
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             1609..1614
                     /codon_start=1
                     /label=Linker w/ AfeI
                     /translation="SA"
     misc_feature    1615
                     /label=frameshift compensation
     misc_feature    1616..1725
                     /label=CRISPIE donor (downstream of insert)
     misc_feature    1616..1625
                     /label=junctional exon
     misc_feature    1626..1627
                     /label=splice donor
     misc_feature    1628..1725
                     /label=junctional intron
     primer_bind     complement(1707..1756)
                     /label=WD0117 Homer1 e8 g8757 RV
     misc_feature    1726..1745
                     /label=targeting sequence
     primer_bind     1726..1745
                     /label=WD0096 Homer1 i17 g8757 gRNA FW
     misc_feature    1746..1748
                     /label=PAM
     misc_feature    1749
                     /label=MluI
     misc_feature    1750..1754
                     /label=Multiple cloning site
     primer_bind     complement(1756..1775)
                     /label=3REV
     repeat_region   1784..1924
                     /label=AAV2 ITR
     primer_bind     complement(1830..1841)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     1885..1902
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      1999..2454
                     /direction=RIGHT
                     /label=f1 ori
     primer_bind     complement(2086..2105)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2296..2317
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2471..2490)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2590..2612
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2650..2668)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2736..2840
                     /label=AmpR promoter
     CDS             2841..3701
                     /codon_start=1
                     /label=AmpR
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3059..3078)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3872..4460
                     /direction=RIGHT
                     /label=ori
     primer_bind     4361..4380
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acggttcctg gccttttgct ggccttttgc tcacatgtat aacttcgtat agtataaatt
       61 atacgaagtt atgatccgac gccgccatct ctaggcccgc gccggccccc tcgcacagac
      121 ttgtgggaga agctcggcta ctcccctgcc ccggttaatt tgcatataat atttcctagt
      181 aactatagag gcttaatgtg cgataaaaga cagataatct gttcttttta atactagcta
      241 cattttacat gataggcttg gatttctata agagatacaa atactaaatt attattttaa
      301 aaaacagcac actcgagaac tcaccctaac tgtaaagtta gtataacttc gtatagtata
      361 aattatacga agttataagc cttgttatat gcctccggtg cccaggtttt agagctagaa
      421 atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
      481 cttttttgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt ttttagcgcg
      541 tgcgccaatt ctgcagacaa atggctctag agctttatat gcctccggtg cccaggggga
      601 gccagggaat aagacgtgaa aggctgtcct gccctggaca ctgggtttgg agctgaggac
      661 tgagcatgtg tgtctggttt tgctttcccc ctctgcagca ggagagcagc tagcggaatg
      721 gcagaaatcg gtactggctt tccattcgac ccccattatg tggaagtcct gggcgagcgc
      781 atgcactacg tcgatgttgg tccgcgcgat ggcacccctg tgctgttcct gcacggtaac
      841 ccgacctcct cctacgtgtg gcgcaacatc atcccgcatg ttgcaccgac ccatcgctgc
      901 attgctccag acctgatcgg tatgggcaaa tccgacaaac cagacctggg ttatttcttc
      961 gacgaccacg tccgcttcat ggatgccttc atcgaagccc tgggtctgga agaggtcgtc
     1021 ctggtcattc acgactgggg ctccgctctg ggtttccact gggccaagcg caatccagag
     1081 cgcgtcaaag gtattgcatt tatggagttc atccgcccta tcccgacctg ggacgaatgg
     1141 ccagaatttg cccgcgagac cttccaggcc ttccgcacca ccgacgtcgg ccgcaagctg
     1201 atcatcgatc agaacgtttt tatcgagggt acgctgccga tgggtgtcgt ccgcccgctg
     1261 actgaagtcg agatggacca ttaccgcgag ccgttcctga atcctgttga ccgcgagcca
     1321 ctgtggcgct tcccaaacga gctgccaatc gccggtgagc cagcgaacat cgtcgcgctg
     1381 gtcgaagaat acatggactg gctgcaccag tcccctgtcc cgaagctgct gttctggggc
     1441 accccaggcg ttctgatccc accggccgaa gccgctcgcc tggccaaaag cctgcctaac
     1501 tgcaaggctg tggacatcgg cccgggtctg aatctgctgc aagaagacaa cccggacctg
     1561 atcggcagcg agatcgcgcg ctggctgtcc acgctcgaga tttccggcag cgctgatcat
     1621 cgaaggtaat ccagtgcgtc tcccaggagg agtggccgtc cagacagcgg ctattaaata
     1681 ccaagacttc aaagtgtcag gagctaggat gctatgatat tattctatat gcctccggtg
     1741 cccaggggac gcgttaactt acggtaaatg gcccggcggc cgcaggaacc cctagtgatg
     1801 gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc
     1861 gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg
     1921 caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     1981 cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     2041 ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct
     2101 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     2161 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg
     2221 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     2281 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg
     2341 gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc
     2401 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt
     2461 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     2521 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     2581 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     2641 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     2701 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     2761 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     2821 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     2881 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     2941 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     3001 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     3061 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     3121 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     3181 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     3241 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     3301 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     3361 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     3421 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     3481 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     3541 gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat ggtaagccct
     3601 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     3661 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     3721 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     3781 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     3841 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     3901 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     3961 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     4021 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     4081 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     4141 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     4201 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     4261 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     4321 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     4381 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     4441 gggggcggag cctatggaaa aacgccagca acgcggcctt ttt
//
