LOCUS       sequence-431465-        3841 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (2A Selection) Violet-excited GFP, T2A, 
            blasticidin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3841)
  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 3841)
  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: AA406
FEATURES             Location/Qualifiers
     source          1..3841
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             444..500
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(501..503,504..1214)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLFIQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(543..564)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(804..823)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1151..1172
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1221..1274
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1275..1670
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(1751..1767)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1751..1767)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1764..1786)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1775..1791
                     /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(1799..1805,1806..1823,1824..1829))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1844..1865
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1982..1999)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2153..2741)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2233..2252)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2919..3728)
                     /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     3642..3661
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3720..3739
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagagaatt cgagctcggt acctcgcgaa
      421 tacatctaga tgaagacgga agcgcaacaa acttctctct gctgaaacaa gccggagatg
      481 tcgaagagaa tcctggaccg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
      541 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
      601 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
      661 ccgtgccctg gcccaccctc gtgaccacct tcagctacgg cgtgcagtgc ttcagccgct
      721 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
      781 aggagcgcac catcagcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
      841 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
      901 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcacgg
      961 ccgacaagca gaagaacggc atcaaggcga acttcaagat ccgccacaac atcgaggacg
     1021 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     1081 tgctgcccga caaccactac ctgttcatcc agtccgccct gagcaaagac cccaacgaga
     1141 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact cacggcatgg
     1201 acgagctgta caagggatcc gagggcaggg gaagccttct aacatgcggg gacgtggagg
     1261 aaaatcccgg ccccgccaag cctttgtctc aagaagaatc caccctcatt gaaagagcaa
     1321 cggctacaat caacagcatc cccatctctg aggactacag cgtcgccagc gcagctctct
     1381 ctagcgacgg ccgcatcttc actggtgtca atgtatatca ttttactggg ggaccttgtg
     1441 cagaactcgt ggtgctgggc actgctgctg ctgcggcagc tggcaacctg acttgtatcg
     1501 tcgcgatcgg aaatgagaac aggggcatct tgagcccctg cggacggtgc cgacaggtgc
     1561 ttctcgatct gcatcctggg atcaaagcca tagtgaagga cagtgatgga cagccgacgg
     1621 cagttgggat tcgtgaattg ctgccctctg gttatgtgtg ggagggctga taaattccag
     1681 tcttcggaac caaatcggat cccgggcccg tcgactgcag aggcctgcat gcaagcttgg
     1741 tgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     1801 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     1861 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     1921 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     1981 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     2041 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     2101 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     2161 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     2221 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     2281 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     2341 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     2401 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     2461 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     2521 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     2581 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     2641 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     2701 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     2761 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     2821 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaagcc
     2881 caatctgaat aatgttacaa ccaattaacc aattctgatt agaaaaactc atcgagcatc
     2941 aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt
     3001 ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat
     3061 cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa
     3121 ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa
     3181 agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa
     3241 tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg
     3301 cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact
     3361 gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct
     3421 gtttttccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc
     3481 ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta
     3541 acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc
     3601 ccatacaagc gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac
     3661 ccatataaat cagcatccat gttggaattt aatcgcggcc tcgacgtttc ccgttgaata
     3721 tggctcataa caccccttgt attactgttt atgtaagcag acagttttat tgttcatgat
     3781 gatatatttt tatcttgtgc aatgtaacat cagagatttt gagacacggg ccagagctgc
     3841 a
//
