LOCUS       sequence-254526-        4436 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  destination vector with N-terminal HA tag .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4436)
  AUTHORS   Hazelbaker DZ, Beccard A, Angelini G, Mazzucato P, Messana A, Lam D,
            Eggan K, Barrett LE
  TITLE     A multiplexed gRNA piggyBac transposon system facilitates efficient 
            induction of CRISPRi and CRISPRa in human pluripotent stem cells.
  JOURNAL   Sci Rep. 2020 Jan 20;10(1):635. doi: 10.1038/s41598-020-57500-1.
  PUBMED    31959800
REFERENCE   2  (bases 1 to 4436)
  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: "Sci Rep."; 
            date: "2020-01-20"; volume: "10(1):635. doi"; pages: " 
            10.1038/s41598-020-57500-1"
COMMENT     Sequence Label: S2_AscI_HA-gateway_Bsu36I
FEATURES             Location/Qualifiers
     source          1..4436
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(28..50)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     150..169
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     363..385
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     377..394
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     378..394
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             478..504
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    505..629
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(654..659,660..677,678..684)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             738..1397
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(804..823)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1739..2044
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1923..1942
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2085..2209)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2259..2275)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2259..2275)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2272..2294)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2283..2299
                     /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(2307..2313,2314..2331,2332..2337))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2352..2373
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2490..2507)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2661..3249)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2741..2760)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3420..4235)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(3420..4235)
                     /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="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4143..4162
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4236..4340)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4408..4426
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
       61 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
      121 tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca
      181 ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgcca
      241 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
      301 acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt
      361 ttcccagtca cgacgttgta aaacgacggc cagtgaattg acgcgtattg ggatggcgcg
      421 cctatcgaaa ttcactatag ggagacccaa gctggctagt taagcttcgc caccatgtac
      481 ccatacgatg ttccagatta cgctacaagt ttgtacaaaa aagctgaacg agaaacgtaa
      541 aatgatataa atatcaatat attaaattag attttgcata aaaaacagac tacataatac
      601 tgtaaaacac aacatatcca gtcactatgg cggccgcatt aggcacccca ggctttacac
      661 tttatgcttc cggctcgtat aatgtgtgga ttttgagtta ggatccggcg agattttcag
      721 gagctaagga agctaaaatg gagaaaaaaa tcactggata taccaccgtt gatatatccc
      781 aatggcatcg taaagaacat tttgaggcat ttcagtcagt tgctcaatgt acctataacc
      841 agaccgttca gctggatatt acggcctttt taaagaccgt aaagaaaaat aagcacaagt
      901 tttatccggc ctttattcac attcttgccc gcctgatgaa tgctcatccg gaattccgta
      961 tggcaatgaa agacggtgag ctggtgatat gggatagtgt tcacccttgt tacaccgttt
     1021 tccatgagca aactgaaacg ttttcatcgc tctggagtga ataccacgac gatttccggc
     1081 agtttctaca catatattcg caagatgtgg cgtgttacgg tgaaaacctg gcctatttcc
     1141 ctaaagggtt tattgagaat atgtttttcg tctcagccaa tccctgggtg agtttcacca
     1201 gttttgattt aaacgtggcc aatatggaca acttcttcgc ccccgttttc accatgggca
     1261 aatattatac gcaaggcgac aaggtgctga tgccgctggc gattcaggtt catcatgccg
     1321 tctgtgatgg cttccatgtc ggcagaatgc ttaatgaatt acaacagtac tgcgatgagt
     1381 ggcagggcgg ggcgtaaacg cgtggatccg gcttactaaa agccagataa cagtatgcgt
     1441 atttgcgcgc tgatttttgc ggtataagaa tatatactga tatgtatacc cgaagtatgt
     1501 caaaaagagg tgtgctatga agcagcgtat tacagtgaca gttgacagcg acagctatca
     1561 gttgctcaag gcatatatga tgtcaatatc tccggtctgg taagcacaac catgcagaat
     1621 gaagcccgtc gtctgcgtgc cgaacgctgg aaagcggaaa atcaggaagg gatggctgag
     1681 gtcgcccggt ttattgaaat gaacggctct tttgctgacg agaacaggga ctggtgaaat
     1741 gcagtttaag gtttacacct ataaaagaga gagccgttat cgtctgtttg tggatgtaca
     1801 gagtgatatt attgacacgc ccgggcgacg gatggtgatc cccctggcca gtgcacgtct
     1861 gctgtcagat aaagtctccc gtgaacttta cccggtggtg catatcgggg atgaaagctg
     1921 gcgcatgatg accaccgata tggccagtgt gccggtctcc gttatcgggg aagaagtggc
     1981 tgatctcagc caccgcgaaa atgacatcaa aaacgccatt aacctgatgt tctggggaat
     2041 ataaatgtca ggctccgtta tacacagcca gtctgcaggt cgaccatagt gactggatat
     2101 gttgtgtttt acagtattat gtagtctgtt ttttatgcaa aatctaattt aatatattga
     2161 tatttatatc attttacgtt tctcgttcag ctttcttgta caaagtggta gtaatgacct
     2221 taggatccca atggcgcgcc gagcttggct cgagcatggt catagctgtt tcctgtgtga
     2281 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     2341 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     2401 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     2461 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     2521 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     2581 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     2641 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     2701 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     2761 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     2821 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     2881 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     2941 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     3001 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     3061 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     3121 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     3181 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     3241 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     3301 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     3361 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     3421 tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata
     3481 ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat
     3541 aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct
     3601 attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact
     3661 gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag
     3721 ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc
     3781 gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa
     3841 tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat
     3901 tcttctaata cctggaatgc tgttttccca gggatcgcag tggtgagtaa ccatgcatca
     3961 tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt
     4021 agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac
     4081 aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca
     4141 ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc
     4201 ctagagcaag acgtttcccg ttgaatatgg ctcatactct tcctttttca atattattga
     4261 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     4321 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     4381 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtct
//
