LOCUS       sequence-431469-        3700 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (2A Selection) Hygromycin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3700)
  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 3700)
  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: AA149
FEATURES             Location/Qualifiers
     source          1..3700
                     /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             453..506
                     /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             507..1532
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVHRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     primer_bind     complement(1610..1626)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1610..1626)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1623..1645)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1634..1650
                     /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(1658..1664,1665..1682,1683..1688))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1703..1724
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1841..1858)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2012..2600)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2092..2111)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2778..3587)
                     /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     3501..3520
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3579..3598
                     /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 agcggcagtg gagagggcag aggaagtctg ctaacatgcg
      481 gtgacgtcga ggagaatcct ggcccaatga aaaagcctga actcaccgcg acgtctgtcg
      541 agaagtttct gatcgaaaag ttcgacagcg tgtccgacct gatgcagctc tcggagggcg
      601 aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata
      661 gctgcgccga tggtttctac aaagatcgtt atgttcatcg gcactttgca tcggccgcgc
      721 tcccgattcc ggaagtgctt gacattgggg aatttagcga gagcctgacc tattgcatct
      781 cccgccgtgc acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc
      841 tgcagccggt cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg
      901 ggttcggccc attcggaccg caaggaatcg gtcaatacac tacgtggcgt gatttcatat
      961 gcgcgattgc tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg
     1021 cgtccgtcgc gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc
     1081 ggcacctcgt gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa
     1141 cagcggtcat tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca
     1201 tcttcttctg gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga
     1261 ggcatccgga gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg
     1321 accaactcta tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc
     1381 gatgcgacgc aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca
     1441 gaagcgcggc cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac
     1501 gccccagcac tcgtccgagg gcaaaggaat aaattccagt cttcggaacc aaatcggatc
     1561 ccgggcccgt cgactgcaga ggcctgcatg caagcttggt gtaatcatgg tcatagctgt
     1621 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     1681 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     1741 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     1801 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     1861 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     1921 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     1981 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     2041 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     2101 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     2161 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     2221 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     2281 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     2341 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     2401 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     2461 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     2521 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     2581 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     2641 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     2701 agatcctttt aaattaaaaa tgaagtttta aatcaagccc aatctgaata atgttacaac
     2761 caattaacca attctgatta gaaaaactca tcgagcatca aatgaaactg caatttattc
     2821 atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac
     2881 tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt
     2941 ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa
     3001 tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag
     3061 acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg
     3121 ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa
     3181 ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt
     3241 tcacctgaat caggatattc ttctaatacc tggaatgctg tttttccggg gatcgcagtg
     3301 gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata
     3361 aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct
     3421 ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaagcg atagattgtc
     3481 gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg
     3541 ttggaattta atcgcggcct cgacgtttcc cgttgaatat ggctcataac accccttgta
     3601 ttactgttta tgtaagcaga cagttttatt gttcatgatg atatattttt atcttgtgca
     3661 atgtaacatc agagattttg agacacgggc cagagctgca
//
