LOCUS       sequence-431466-        4165 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (CROPseq) WPRE;  3'LTR; guide expression bGHpolyA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4165)
  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 4165)
  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: AA269
FEATURES             Location/Qualifiers
     source          1..4165
                     /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             1118..1298
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(1324..1341)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1330..1554
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1654..1670)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1654..1670)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1667..1689)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1678..1694
                     /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(1702..1708,1709..1726,1727..1732))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1747..1768
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1885..1902)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2056..2644)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2136..2155)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2822..3631)
                     /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     3545..3564
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3623..3642
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(3773..3795)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3895..3914
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4108..4130
                     /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 ttcgtctcag tttaagagct atgctggaaa cagcatagca agtttaaata
     1021 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt taagcttggc
     1081 gtaactagat cttgagacac tgctttttgc ttgtactggg tctctctggt tagaccagat
     1141 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
     1201 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
     1261 cctcagaccc ttttagtcag tgtggaaaat ctctagcagg gcccgtttaa acccgctgat
     1321 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     1381 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     1441 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     1501 gggaggattg ggaagataat agcaggcatg ctggggatgc ggtgggctct atggcttctg
     1561 aggcggaaag aaccagcgtc cagtcttcgg aaccaaatcg gatcccgggc ccgtcgactg
     1621 cagaggcctg catgcaagct tggtgtaatc atggtcatag ctgtttcctg tgtgaaattg
     1681 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     1741 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     1801 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     1861 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     1921 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     1981 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     2041 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     2101 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     2161 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     2221 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     2281 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     2341 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     2401 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     2461 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     2521 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     2581 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     2641 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     2701 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     2761 aaaatgaagt tttaaatcaa gcccaatctg aataatgtta caaccaatta accaattctg
     2821 attagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
     2881 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
     2941 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
     3001 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
     3061 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
     3121 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
     3181 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
     3241 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     3301 attcttctaa tacctggaat gctgtttttc cggggatcgc agtggtgagt aaccatgcat
     3361 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     3421 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     3481 acaactctgg cgcatcgggc ttcccataca agcgatagat tgtcgcacct gattgcccga
     3541 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     3601 gcctcgacgt ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag
     3661 cagacagttt tattgttcat gatgatatat ttttatcttg tgcaatgtaa catcagagat
     3721 tttgagacac gggccagagc tgcatcgcgc gtttcggtga tgacggtgaa aacctctgac
     3781 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag
     3841 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat
     3901 cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa
     3961 ggagaaaata ccgcatcagg cgccattcgc cattcaggct gcgcaactgt tgggaagggc
     4021 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc
     4081 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagag
     4141 aattcgagct cggtacctcg cgaat
//
