LOCUS       sequence-151052-        7496 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  QF2 HACK backbone.  Contains QF2-hsp70, but no gRNAs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7496)
  AUTHORS   Lin CC, Potter CJ
  TITLE     Editing Transgenic DNA Components by Inducible Gene Replacement in 
            Drosophila melanogaster.
  JOURNAL   Genetics. 2016 Jun 22. pii: genetics.116.191783.
  PUBMED    27334272
REFERENCE   2  (bases 1 to 7496)
  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: "Genetics. 
            2016 Jun 22. pii: genetics.116.191783."
COMMENT     Sequence Label: pHACK-QF2
FEATURES             Location/Qualifiers
     source          1..7496
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     622..641
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(860..951)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     993..1012
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1018..1037
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    1244..1277
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     protein_bind    1291..1324
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     CDS             1488..1541
                     /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"
     protein_bind    complement(2874..2907)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    2971..3092
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2982..3001)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3036..3055
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3212..3922)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     3759..3777
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     protein_bind    complement(4100..4133)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4156..4952
                     /label=dU6-1 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-1 
                     snRNA (Port et al., 2014)"
     misc_RNA        4971..5046
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        5047..5467
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     misc_RNA        5486..5561
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(5994..6027)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     primer_bind     complement(6497..6514)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6497..6513)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6612..6629)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6783..7371)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6863..6882)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7495..7496,1..790,791..859))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
       61 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
      121 gcgctgcgat gataccgcga gaaccacgct caccggctcc ggatttatca gcaataaacc
      181 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
      241 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
      301 ttgttgccat cgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
      361 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
      421 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
      481 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
      541 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
      601 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
      661 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
      721 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
      781 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
      841 ggaaatgttg aatactcata ttcttccttt ttcaatatta ttgaagcatt tatcagggtt
      901 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggtca
      961 gtgttacaac caattaacca attctgaaca ttatcgcgag cccatttata cctgaatatg
     1021 gctcataaca ccccttgttt gcctggcggc agtagcgcgg tggtcccacc tgaccccatg
     1081 ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg tggggactcc ccatgcgaga
     1141 gtagggaact gccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg
     1201 cccgggctaa ttatggggtg tcgcccttat tcgactctat agtgaagttc ctattctcta
     1261 gaaagtatag gaacttctga agtggggcgg gtgccagggc gtgcccttgg gctccccggg
     1321 cgcgtacaag ccgaaggcgc gcatgatgaa ataacataag gtggtcccgt cgaaagccga
     1381 agcttaccga agtatacact taaattcagt gcacgtttgc ttgttgagag gaaaggttgt
     1441 gtgcggacga attttttttt gaaaacatta acccttacgt aacgcgtgag ggccgcggca
     1501 gcctgctgac ctgcggcgat gtggaggaga accccgggcc ccctaggatg ccacccaagc
     1561 gcaaaacgct taacgctgcg gctgaggcta acgctcatgc cgacggacac gccgacggaa
     1621 acgccgacgg acacgtggcc aatacggccg cgtcctcgaa taatgcgagg ttcgctgatc
     1681 tcactaacat cgatactccg ggtctgggac ccacaactac gaccctgctc gtggaaccag
     1741 cacgctcaaa gcgtcaacga gtgtcccgcg catgcgacca gtgccgtgca gcccgagaga
     1801 aatgcgacgg aatacagcct gcgtgtttcc cgtgcgtttc ccagggaagg tcctgcactt
     1861 atcaggcttc gccgaaaaag aggggagttc aaaccggtta tattcgtacg ctggagctcg
     1921 ccctcgcctg gatgtttgaa aatgtcgcgc gttccgaaga tgccttgcat aacctcctcg
     1981 tccgtgacgc cggacaagga tcagctctgc tcgttggtaa agattcgccg gctgccgagc
     2041 gactccatgc ccgttgggct actagccgtg tcaataagag cattacccgc ctcctccgtc
     2101 agttggagct ccctcctacc gccacggcta cggcctcgat aatgccgcac gtgatggagc
     2161 agcctctcag taccagcatt aaccccgtca acgaccgctt caacggtatt cccaacccca
     2221 ctccgtataa ctccgatgca gctctcgatg ctatcactca gaccaacgat tatggaagcg
     2281 taaatacaca tggtatcctc tctacttacc cgccaccggc tacgcacctt aatgaagctt
     2341 ccgtcgctct cgctcccggt ggcgcccccc cccgaccgcc tcctccgtat gttgacagca
     2401 cgaccaatca cccgccgtac cactcgaatc tggttccaat ggcgaacttt ggttactcga
     2461 ccgttgatta cgatgccatg gttgacgatt tggctagcat tgaatacacg gacgctgtgg
     2521 atgtcgaccc acagtttatg accaatctgg gattcgttcc tggatgtaac ttctccgaca
     2581 ttaatacata cgaacagtga tgacgtctat cgataccgtc gactaaagcc aaatagaaaa
     2641 ttattcagtt cctggcttaa gtttttaaaa gtgatattat ttatttggtt gtaaccaacc
     2701 aaaagaatgt aaataactaa tacataatta tgttagtttt aagttagcaa caaattgatt
     2761 ttagctatat tagctacttg gttaataaat agaatatatt tatttaaaga taattgcgtt
     2821 tttattgtca gggagtgagt ttgcttaaaa actcgtttag atccaccgca tgcataactt
     2881 cgtatagcat acattatacg aagttatggt accactcgac gattaaccgt tagtttagca
     2941 agtataattg aattttttat aagatccagc taagatacat tgatgagttt ggacaaacca
     3001 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
     3061 ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt
     3121 ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg
     3181 gtatggctga ttatgatcag agtcgcggcg cttacttgta cagctcgtcc atgccgccgg
     3241 tggagtggcg gccctcggcg cgttcgtact gttccacgat ggtgtagtcc tcgttgtggg
     3301 aggtgatgtc caacttgatg ttgacgttgt aggcgccggg cagctgcacg ggcttcttgg
     3361 ccttgtaggt ggtcttgacc tcagcgtcgt agtggccgcc gtccttcagc ttcagcctct
     3421 gcttgatctc gcccttcagg gcgccgtcct cggggtacat ccgctcggag gaggcctccc
     3481 agcccatggt tttcttctgc attacggggc cgtcggaggg gaagttggtg ccgcgcagct
     3541 tcaccttgta gatgaactcg ccgtcctgca gggaggagtc ctgggtcacg gtcaccacgc
     3601 cgccgtcctc gaagttcatc acgcgctccc acttgaagcc ctcggggaag gacagcttca
     3661 agtagtcggg gatgtcggcg gggtgcttca cgtaggcctt ggagccgtac atgaactgag
     3721 gggacaggat gtcccaggcg aagggcaggg ggccaccctt ggtcaccttc agcttggcgg
     3781 tctgggtgcc ctcgtagggg cggccctcgc cctcgccctc gatctcgaac tcgtggccgt
     3841 tcacggagcc ctccatgtgc accttgaagc gcatgaactc cttgatgatg gccatgttat
     3901 cctcctcgcc cttgctcacc atgggccgat tgtttagctt gttcagctgc gcttgtttat
     3961 ttgcttagct ttcgcttagc gacgtgttca ctttgcttgt ttgaattgaa ttgtcgctcc
     4021 gtagacgaag cgcctctatt tatactccgg cgctaattga attagctcta attgaattag
     4081 tctctaattg aatgacgtca taacttcgta tagcatacat tatacgaagt tatagatcac
     4141 tagtgatctg aattcatttt caacgtcctc gatagtatag tggttagtat ccccgcctgt
     4201 cacgcgggag accggggttc aattccccgt cggggagaat ctgtgattct tttttttttt
     4261 cttttacttt gttatataaa caatttttgt tttaattgaa tctaatttgc cattgctttt
     4321 aggaatctca ggcatccagc aagcgtttgt ccgccgaatc gcccatcagt gaagaagatc
     4381 ctgtggcggc tacgaaaatc tccccggcca tgtcggcctc cacctccagc gaaaaaccca
     4441 tcagcgagct ggccacctct gtgctgaccc accgctttcc agactccacc tcctcacccg
     4501 gcgaacatgg ccttggacga atgcagttgt cgatccgcta cagcgcccag cgtcaaaaac
     4561 tagacgtgac catacacaaa atccagaaga taccacttcg cgatcccagc aatatccccg
     4621 atccgtatgt taagctgtat ctgttgcctg gacgcaccaa ggagtcgaaa cgcaagacga
     4681 gcgtgatcaa ggacaactgc aaccccgtct acgatgcatc ctttgagtac ctgatttcca
     4741 ttgccgaact caggcagacg gaactggagg tgacggtgtg cacccaaaag ggattcctat
     4801 ccggcggtag tcccatcatt ggcatggtag gtacccgaaa gcaacccctt agttacagac
     4861 acagcgcgta cgtccttcgc atccttatga ttcccaagta catattctgc aagagtacag
     4921 tatatatagg aaagatatcc gggtgaactt cggggtcttc gagaagacct gttttagagc
     4981 tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt
     5041 cggtgctttt ttgctcacct gtgattgctc ctactcaaat acaaaaacat caaattttct
     5101 gtcaataaag catatttatt tatatttatt ttacaggaaa gaattccttt taaagtgtat
     5161 tttaacctat aatgaaaaac gattaaaaaa aatacataaa ataattcgaa aatttttgaa
     5221 tagcccaggt tgataaaaat tcatttcata cgttttataa cttatgcccc taagtatttt
     5281 ttgaccatag tgtttcaatt ctacattaat tttacagagt agaatgaaac gccacctact
     5341 cagccaagag gcgaaaaggt tagctcgcca agcagagagg gcgccagtgc tcactacttt
     5401 ttataattct caacttcttt ttccagactc agttcgtata tatagaccta ttttcaattt
     5461 aacgtcgggg tcttcgagaa gacctgtttt agagctagaa atagcaagtt aaaataaggc
     5521 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttgcc tacctggagc
     5581 ctgagagttg ttcaataaaa taaaaatgtt tcgttttttt gctttcgcca gtatttatta
     5641 tttttcatca atatgtattc aatttggtat gtatttagta attgtaatat atagacaatg
     5701 gttttccgtt gacgtacata catctgacgt gtgtttattt agacataata gttatgtttt
     5761 cacatctttt taatgttcgc ttaatgcgta tgcttaatta attctgacgt aagctagtta
     5821 caaaccccac ggacatgcta agggttaatc aacaatcata tcgctgtctc actcagactc
     5881 aatacgacac tcagaatact attcctttca ctcgcactta ttgcaagcat acgttaagtg
     5941 gatgtctctt gccgacggga ccaccttatg ttatttcatc atgaaaatga agtgaagttc
     6001 ctatactttc tagagaatag gaacttctat agtgagtcga ataagggcga cacaaaattt
     6061 attctaaatg cataataaat actgataaca tcttatagtt tgtattatat tttgtattat
     6121 cgttgacatg tataattttg atatcaaaaa ctgattttcc ctttattatt ttcgagattt
     6181 attttcttaa ttctctttaa caaactagaa atattgtata tacaaaaaat cataaataat
     6241 agatgaatag tttaattata ggtgttcatc aatcgaaaaa gcaacgtatc ttatttaaag
     6301 tgcgttgctt ttttctcatt tataaggtta aataattctc atatatcaag caaagtgaca
     6361 ggcgccctta aatattctga caaatgctct ttccctaaac tccccccata aaaaaacccg
     6421 ccgaagcggg tttttacgtt atttgcggat taacgattac tcgttatcag aaccgcccag
     6481 ggggcccgag cttaagactg gccgtcgttt tacaacacag aaagagtttg tagaaacgca
     6541 aaaaggccat ccgtcagggg ccttctgctt agtttgatgc ctggcagttc cctactctcg
     6601 ccttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     6661 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     6721 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     6781 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     6841 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     6901 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     6961 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     7021 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     7081 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     7141 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     7201 gctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     7261 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     7321 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     7381 ttgatctttt ctacggggtc tgacgctcag tggaacgacg cgcgcgtaac tcacgttaag
     7441 ggattttggt catgagcttg cgccgtcccg tcaagtcagc gtaatgctct gctttt
//
