LOCUS       MIGR1_U6gRNA1_PG        7370 bp    DNA     circular SYN 01-MAR-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7370)
  AUTHORS   City of Hope
  TITLE     Direct Submission
  JOURNAL   Exported Mar 28, 2025 from SnapGene 8.0.3
            https://www.snapgene.com
COMMENT     Sequence Label: MIGR1-U6gRNA1-PGK-TagBFP_v2_filler
FEATURES             Location/Qualifiers
     source          1..7370
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             123..639
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    703..1044
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             1111..1527
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1457..1479
                     /label=LXSN F
     primer_bind     1518..1564
                     /label=U6g1 F
     promoter        1546..1786
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_feature    1803..2419
                     /label=filler
     misc_RNA        2428..2503
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(2487..2543)
                     /label=U6g2 R4
     misc_feature    complement(2490..2510)
                     /label=gib_sgRNAlib_rev_scaffold
     primer_bind     complement(2493..2532)
                     /label=U6g1 R4
     primer_bind     2523..2573
                     /label=PGK F2
     misc_feature    complement(2535..2555)
                     /label=gib_sgRNAlib_rev_overlapping
     misc_feature    2550..3049
                     /label=PGK promoter
     regulatory      3068..3073
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3074..3775
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     primer_bind     complement(3750..3772)
                     /label=TagBFP R4
     primer_bind     complement(3756..3818)
                     /label=TagBFP R1
     LTR             3887..4401
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4563..4579
                     /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)."
     promoter        complement(join(4587..4593,4594..4611,4612..4617))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4632..4653
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4941..5529)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5700..6491,6492..6560))
                     /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(6561..6665)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaagacagga tatcagtggt ccaggctcta gttttgactc aacaatatca ccagctgaag
       61 cctatagagt acgagccata gataaaataa aagattttat ttagtctcca gaaaaagggg
      121 ggaatgaaag accccacctg taggtttggc aagctagctt aagtaacgcc attttgcaag
      181 gcatggaaaa tacataactg agaatagaga agttcagatc aaggttagga acagagagac
      241 agcagaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc
      301 aagaacagat ggtccccaga tgcggtcccg ccctcagcag tttctagaga accatcagat
      361 gtttccaggg tgccccaagg acctgaaatg accctgtgcc ttatttgaac taaccaatca
      421 gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa agagcccaca
      481 acccctcact cggcgcgcca gtcctccgat agactgcgtc gcccgggtac ccgtattccc
      541 aataaagcct cttgctgttt gcatccgaat cgtggactcg ctgatccttg ggagggtctc
      601 ctcagattga ttgactgccc acctcggggg tctttcattt ggaggttcca ccgagatttg
      661 gagacccctg cccagggacc accgaccccc ccgccgggag gtaagctggc cagcggtcgt
      721 ttcgtgtctg tctctgtctt tgtgcgtgtt tgtgccggca tctaatgttt gcgcctgcgt
      781 ctgtactagt tagctaacta gctctgtatc tggcggaccc gtggtggaac tgacgagttc
      841 tgaacacccg gccgcaaccc tgggagacgt cccagggact ttgggggccg tttttgtggc
      901 ccgacctgag gaagggagtc gatgtggaat ccgaccccgt caggatatgt ggttctggta
      961 ggagacgaga acctaaaaca gttcccgcct ccgtctgaat ttttgctttc ggtttggaac
     1021 cgaagccgcg cgtcttgtct gctgcagcgc tgcagcatcg ttctgtgttg tctctgtctg
     1081 actgtgtttc tgtatttgtc tgaaaattag ggccagactg ttaccactcc cttaagtttg
     1141 accttaggtc actggaaaga tgtcgagcgg atcgctcaca accagtcggt agatgtcaag
     1201 aagagacgtt gggttacctt ctgctctgca gaatggccaa cctttaacgt cggatggccg
     1261 cgagacggca cctttaaccg agacctcatc acccaggtta agatcaaggt cttttcacct
     1321 ggcccgcatg gacacccaga ccaggtcccc tacatcgtga cctgggaagc cttggctttt
     1381 gacccccctc cctgggtcaa gccctttgta caccctaagc ctccgcctcc tcttcctcca
     1441 tccgccccgt ctctccccct tgaacctcct cgttcgaccc cgcctcgatc ctccctttat
     1501 ccagccctca ctccttctct aggcgccgga attagatctc tcgaggaggg cctatttccc
     1561 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tagaattaat
     1621 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
     1681 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
     1741 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgggtct
     1801 tcgcaatttg agttaagggt gttgctctca atgatttcat taatggttca atttttaatt
     1861 tcttttcttc tggtttaaaa ttcaagttta aagtgaaagt gtaatatgca cccatttctt
     1921 taaataaatc ttctaaatag tctactaatg ttttattttg ttttttataa aatcaagcag
     1981 cctctgctat taatatagaa gcttgtattc catctttatc tctagctgag tcatcaatta
     2041 catatccata actttcttca taagcaaaaa caaaatttaa tccgttatct tcttctttag
     2101 caatttctct acccattcat ttaaatccag ttaaagtttt tacaatatta actccatatt
     2161 tttcatgagc gattctatca cccaaatcac ttgttacaaa acttgaatat agagccggat
     2221 tttttggaat gctatttaag cgttttagat ttgataattt tcaatcaatt aaaattggtc
     2281 ctgtttgatt tccatctaat cttacaaaat gaccatcatg ttttattgcc attccaaatc
     2341 tgtcagcatc tgggtcattc ataataataa tatctgcatc atgtttaata ccatattcaa
     2401 gcggtatttt tcatgcaggg aagaccagtt ttagagctag aaatagcaag ttaaaataag
     2461 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt gcacagcgcg
     2521 tatgtgaatt cggtggatcc aattctaccg ggtaggggag gcgcttttcc caaggcagtc
     2581 tggagcatgc gctttagcag ccccgctggg cacttggcgc tacacaagtg gcctctggcc
     2641 tcgcacacat tccacatcca ccggtaggcg ccaaccggct ccgttctttg gtggcccctt
     2701 cgcgccacct tctactcctc ccctagtcag gaagttcccc cccgccccgc agctcgcgtc
     2761 gtgcaggacg tgacaaatgg aagtagcacg tctcactagt ctcgtgcaga tggacagcac
     2821 cgctgagcaa tggaagcggg taggcctttg gggcagcggc caatagcagc tttgctcctt
     2881 cgctttctgg gctcagaggc tgggaagggg tgggtccggg ggcgggctca ggggcgggct
     2941 caggggcggg gcgggcgccc gaaggtcctc cggaggcccg gcattctgca cgcttcaaaa
     3001 gcgcacgtct gccgcgctgt tctcctcttc ctcatctccg ggcctttcgg accggttcta
     3061 gagcgctgcc accatgagcg agctgattaa ggagaacatg cacatgaagc tgtacatgga
     3121 gggcaccgtg gacaaccatc acttcaagtg cacatccgag ggcgaaggca agccctacga
     3181 gggcacccag accatgagaa tcaaggtggt cgagggcggc cctctcccct tcgccttcga
     3241 catcctggct actagcttcc tctacggcag caagaccttc atcaaccaca cccagggcat
     3301 ccccgacttc ttcaagcagt ccttccctga gggcttcaca tgggagagag tcaccacata
     3361 cgaggacggg ggcgtgctga ccgctaccca ggacaccagc ctccaggacg gctgcctcat
     3421 ctacaacgtc aagatcagag gggtgaactt cacatccaac ggccctgtga tgcagaagaa
     3481 aacactcggc tgggaggcct tcaccgagac gctgtacccc gctgacggcg gcctggaagg
     3541 cagaaacgac atggccctga agctcgtggg cgggagccat ctgatcgcaa acatcaagac
     3601 cacatataga tccaagaaac ccgctaagaa cctcaagatg cctggcgtct actatgtgga
     3661 ctacagactg gaaagaatca aggaggccaa caacgagacc tacgtcgagc agcacgaggt
     3721 ggcagtggcc agatactgcg acctccctag caaactgggg cacaagctta attgagcggc
     3781 cgctgaatta attaagaatt atcaagctta tcgataccgt cgactcctgc agccaagctt
     3841 atcgataaaa taaaagattt tatttagtct ccagaaaaag gggggaatga aagaccccac
     3901 ctgtaggttt ggcaagctag cttaagtaac gccattttgc aaggcatgga aaatacataa
     3961 ctgagaatag agaagttcag atcaaggtta ggaacagaga gacagcagaa tatgggccaa
     4021 acaggatatc tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggtcccc
     4081 agatgcggtc ccgccctcag cagtttctag agaaccatca gatgtttcca gggtgcccca
     4141 aggacctgaa atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct
     4201 gttcgcgcgc ttctgctccc cgagctcaat aaaagagccc acaacccctc actcggcgcg
     4261 ccagtcctcc gatagactgc gtcgcccggg tacccgtgta tccaataaac cctcttgcag
     4321 ttgcatccga cttgtggtct cgctgttcct tgggagggtc tcctctgagt gattgactac
     4381 ccgtcagcgg gggtctttca tgggtaacag tttcttgaag ttggagaaca acattctgag
     4441 ggtaggagtc gaatattaag taatcctgac tcaattagcc actgttttga atccacatac
     4501 tccaatactc ctgaaatagt tcattatgga cagcgcagaa agagctgggg agaattgtga
     4561 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     4621 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     4681 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4741 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4801 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4861 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4921 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4981 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5041 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5101 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     5161 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5221 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5281 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5341 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     5401 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5461 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     5521 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     5581 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     5641 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5701 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     5761 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     5821 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     5881 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     5941 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     6001 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     6061 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     6121 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     6181 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     6241 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     6301 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     6361 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     6421 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     6481 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     6541 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     6601 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     6661 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
     6721 ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac
     6781 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat
     6841 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg
     6901 cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata
     6961 ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc attcgccatt caggctgcgc
     7021 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg
     7081 ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt
     7141 aaaacgacgg cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca
     7201 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
     7261 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
     7321 tgatgccggc cacgatgcgt ccggcgtaga ggcgattagt ccaatttgtt
//
