LOCUS       sequence-431520-        4155 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (CROPseq) guide expression; UMI-compatible.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4155)
  AUTHORS   McGee AV, Liu YV, Griffith AL, Szegletes ZM, Wen B, Kraus C, Miller 
            NW, Steger RJ, Escude Velasco B, Bosch JA, Zirin JD, Viswanatha R, 
            Sontheimer EJ, Goodale A, Greene MA, Green TM, Doench JG
  TITLE     Modular vector assembly enables rapid assessment of emerging CRISPR 
            technologies.
  JOURNAL   Cell Genom. 2024 Mar 13;4(3):100519. doi: 
            10.1016/j.xgen.2024.100519.
  PUBMED    38484704
REFERENCE   2  (bases 1 to 4155)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell 
            Genom."; date: "2024-03-13"; volume: "4(3):100519. doi"; pages: " 
            10.1016/j.xgen.2024.100519"
COMMENT     Sequence Label: AA271
FEATURES             Location/Qualifiers
     source          1..4155
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    23..611
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(76..96)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(494..505)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        710..958
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     710..730
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     881..900
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     LTR             1108..1288
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(1314..1331)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1320..1544
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1644..1660)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1644..1660)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1657..1679)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1668..1684
                     /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(1692..1698,1699..1716,1717..1722))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1737..1758
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1875..1892)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2046..2634)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2126..2145)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2812..3621)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3535..3554
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3613..3632
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(3763..3785)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3885..3904
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4098..4120
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 acatctagat gaagacggat tcaatcaacc tctggattac aaaatttgtg aaagattgac
       61 tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt
      121 gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt
      181 gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt
      241 gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg
      301 gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg
      361 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc
      421 atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt
      481 ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc
      541 tctgcggcct cttccgcgtc ctcgccttcg ccctcagacg agtcggatct ccctttgggc
      601 cgcctccccg cgtcggctag cacactggaa gggctaattc actcccaacg aagataagat
      661 ttcaccatta tcgtttcaga cccacctccc aaccccgagg ggacccagag agggcctatt
      721 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattagaat
      781 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt
      841 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt
      901 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgg
      961 agacgctttt ttgtttgaga gctagaaacg tctcatagca agttcaaata aggctagtcc
     1021 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt taagcttggc gtaactagat
     1081 cttgagacac tgctttttgc ttgtactggg tctctctggt tagaccagat ctgagcctgg
     1141 gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg
     1201 cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc
     1261 ttttagtcag tgtggaaaat ctctagcagg gcccgtttaa acccgctgat cagcctcgac
     1321 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     1381 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     1441 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     1501 ggaagataat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     1561 aaccagcgtc cagtcttcgg aaccaaatcg gatcccgggc ccgtcgactg cagaggcctg
     1621 catgcaagct tggtgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     1681 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     1741 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     1801 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     1861 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     1921 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     1981 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     2041 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     2101 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     2161 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     2221 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     2281 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     2341 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     2401 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     2461 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     2521 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     2581 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     2641 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     2701 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     2761 tttaaatcaa gcccaatctg aataatgtta caaccaatta accaattctg attagaaaaa
     2821 ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt
     2881 ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc
     2941 aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt
     3001 cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg
     3061 tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg
     3121 ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc
     3181 gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg
     3241 gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa
     3301 tacctggaat gctgtttttc cggggatcgc agtggtgagt aaccatgcat catcaggagt
     3361 acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac
     3421 catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg
     3481 cgcatcgggc ttcccataca agcgatagat tgtcgcacct gattgcccga cattatcgcg
     3541 agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg gcctcgacgt
     3601 ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag cagacagttt
     3661 tattgttcat gatgatatat ttttatcttg tgcaatgtaa catcagagat tttgagacac
     3721 gggccagagc tgcatcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
     3781 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
     3841 cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat
     3901 tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata
     3961 ccgcatcagg cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     4021 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg
     4081 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagag aattcgagct
     4141 cggtacctcg cgaat
//
