LOCUS       sequence-82318-e        5114 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of FRT5-flanked Neo; homologous recombination targeting 
            vector cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5114)
  AUTHORS   Tasic B, Hippenmeyer S, Wang C, Gamboa M, Zong H, Chen-Tsai Y, Luo L
  TITLE     Site-specific integrase-mediated transgenesis in mice via pronuclear
            injection.
  JOURNAL   Proc Natl Acad Sci U S A. 2011 May 10;108(19):7902-7. doi: 
            10.1073/pnas.1019507108. Epub 2011 Apr 4.
  PUBMED    21464299
REFERENCE   2  (bases 1 to 5114)
  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"; doi: 
            "10.1073/pnas.1019507108"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2011-05-10- 10"; volume: "108"; issue: "19"; pages: "7902-7"
COMMENT     Sequence Label: pBT305_pBS-U-attPx3-FRT5-pSV40-Neo-pA-PL-FRT5
FEATURES             Location/Qualifiers
     source          1..5114
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(33..461)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(143..164)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     355..374
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     588..610
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     602..619
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     603..619
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    961..1008
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     1033..1052
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(1240..2034)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(1240..2034)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1351..1370)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1961..1980
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2069..2426)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2083..2218
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        2235..2426
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     2383..2403
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(2428..2532)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        complement(2929..2947)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2968..2984)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2968..2984)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2981..3003)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2992..3008
                     /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(3016..3022,3023..3040,3041..3046))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3061..3082
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3199..3216)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3370..3958)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3450..3469)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4129..4920,4921..4989))
                     /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"
     primer_bind     4752..4771
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4990..5094)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
       61 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
      121 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
      181 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
      241 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
      301 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
      361 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
      421 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct
      481 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
      541 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
      601 ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tgggtacagg
      661 cgcgcccctt actaggcggc cgcgaagttc ctataccttt tgaagaatag gaacttcgga
      721 ataggaactt ctctaggacg tcttaattgt taacttaatt cctgcaggct agagatgatc
      781 ctgagtaacc tgaggctatg gcagggcctg ccgccccgac gttggctgcg agccctgggc
      841 cttcacccga acttgggggg tggggtgggg aaaaggaaga aacgcgggcg tattggcccc
      901 aatggggtct cggtggggta tcgacagagt gccagccctg ggaccgaacc ccgcgtttat
      961 gaacaaacga cccaacaccg tgcgttttat tctgtctttt tattgccgtc atagcgcggg
     1021 ttccttccgg tattgtctcc ttccgtgttt cagttagcct ccccctaggg tgggcgaaga
     1081 actccagcat gagatccccg cgctggagga tcatccagcc ggcgtcccgg aaaacgattc
     1141 cgaagcccaa cctttcatag aaggcggcgg tggaatcgaa atctcgtgat ggcaggttgg
     1201 gcgtcgcttg gtcggtcatt tcgaacccca gagtcccgct cagaagaact cgtcaagaag
     1261 gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg
     1321 gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg ctatgtcctg
     1381 atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc ggccattttc
     1441 caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct cgccgtcggg
     1501 catgctcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat gctcttcgtc
     1561 cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct cgatgcgatg
     1621 tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc
     1681 atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga gatcctgccc
     1741 cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt cgagcacagc
     1801 tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt cttgcagttc
     1861 attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct gcgctgacag
     1921 ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat agccgaatag
     1981 cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa tcatgcgaaa
     2041 cgatcctcat cctgtctctt gatcgatctt tgcaaaagcc taggcctcca aaaaagcctc
     2101 ctcactactt ctggaatagc tcagaggccg aggcggcctc ggcctctgca taaataaaaa
     2161 aaattagtca gccatggggc ggagaatggg cggaactggg cggagttagg ggcgggatgg
     2221 gcggagttag gggcgggact atggttgctg actaattgag atgcatgctt tgcatacttc
     2281 tgcctgctgg ggagcctggg gactttccac acctggttgc tgactaattg agatgcatgc
     2341 tttgcatact tctgcctgct ggggagcctg gggactttcc acaccctaac tgacacacat
     2401 tccacagctg gttctttccg cctcaggact cttccttttt caatattatt gaagcattta
     2461 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     2521 aggggttccg cgcacatttc cccgaaaagt gccacctgaa taatcgctag cgaagttcct
     2581 ataccttttg aagaatagga acttcggaat aggaacttcg tcgacaaagg atccgtcggc
     2641 gaccctacgc ccccaactga gagaactcaa aggttacccc agttggggca ctactcccga
     2701 aaacgtcggc gaccctacgc ccccaactga gagaactcaa aggttacccc agttggggca
     2761 ctactcccga aaacgtcggc gaccctacgc ccccaactga gagaactcaa aggttacccc
     2821 agttggggca ctactcccga aaacgatatc cttacggaat accacttgcc acctatcacc
     2881 agatctggat gcatgatctc ctatttaaat cagctccagc ttttgttccc tttagtgagg
     2941 gttaatttcg agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     3001 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     3061 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     3121 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     3181 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     3241 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     3301 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     3361 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     3421 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     3481 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     3541 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     3601 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     3661 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     3721 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     3781 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
     3841 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     3901 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     3961 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     4021 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     4081 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     4141 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     4201 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     4261 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     4321 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     4381 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     4441 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     4501 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     4561 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     4621 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     4681 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     4741 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     4801 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     4861 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     4921 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     4981 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     5041 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     5101 tccccgaaaa gtgc
//
