LOCUS       sequence-316185-        4834 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Minimal piggyBac ITR-flanked pPgk-pEM7-Neo*-bpA positive selection 
            cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4834)
  AUTHORS   Nam HS, Capecchi MR
  TITLE     Additional gene targeting vector construction plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4834)
  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: 
            "Unpublished"
COMMENT     Sequence Label: PB Neo* PB
FEATURES             Location/Qualifiers
     source          1..4834
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..500
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(36..59)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     433..452
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        512..559
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             578..1381
                     /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="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(641..660)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1251..1270
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1413..1430)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1419..1643
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   1701..1735
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1759..1775)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1759..1775)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        complement(1812..1830)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1851..1867)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1851..1867)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1864..1886)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1875..1891
                     /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(1899..1905,1906..1923,1924..1929))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1944..1965
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2082..2099)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2253..2841)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2333..2352)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3012..3803,3804..3872))
                     /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     3635..3654
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3873..3977)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4003..4458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4140..4161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4352..4371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4585..4607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4599..4616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4600..4616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4623..4642
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4623..4641
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     4666..4682
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     4667..4683
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   4709..4771
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca gccccgctgg
       61 gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc accggtaggc
      121 gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct cccctagtca
      181 ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac
      241 gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg gtaggccttt
      301 ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg ctgggaaggg
      361 gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct
      421 ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt
      481 cctcatctcc gggcctttcg acctgcagcc tgttgacaat taatcatcgg catagtatat
      541 cggcatagta taatacgaca aggtgaggaa ctaaaccatg ggatcggcca ttgaacaaga
      601 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
      661 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
      721 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc
      781 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
      841 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
      901 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
      961 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     1021 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     1081 cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg atgatctcgt
     1141 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     1201 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     1261 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     1321 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     1381 aggggatcaa ttctctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc
     1441 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     1501 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     1561 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     1621 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctcgactag
     1681 agcttgcgga acccttcgaa catgcgtcaa ttttacgcag actatctttc tagggttaaa
     1741 tcagtcaggt acataatgga tccactagtt ctagagcggc cgccaccgcg gtggagctcc
     1801 agcttttgtt ccctttagtg agggttaatt tcgagcttgg cgtaatcatg gtcatagctg
     1861 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     1921 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     1981 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     2041 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     2101 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     2161 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     2221 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     2281 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     2341 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     2401 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     2461 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     2521 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     2581 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     2641 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     2701 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     2761 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     2821 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     2881 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     2941 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     3001 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     3061 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     3121 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     3181 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     3241 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     3301 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     3361 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     3421 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     3481 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     3541 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     3601 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     3661 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     3721 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     3781 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     3841 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     3901 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     3961 caaatagggg ttccgcgcac atttccccga aaagtgccac ctaaattgta agcgttaata
     4021 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     4081 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     4141 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     4201 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     4261 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     4321 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     4381 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     4441 cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg caactgttgg gaagggcgat
     4501 cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat
     4561 taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgaat
     4621 tgtaatacga ctcactatag ggcgaattgg gtaccgggcc ccccctcgag gtcgacggta
     4681 tcgataagct tgatatcgaa ttccttaacc ctagaaagat aatcatattg tgacgtacgt
     4741 taaagataat catgcgtaaa attgacgcat gaggtctgaa gaggagttta cgtccagcca
     4801 agctagcttg gctgcaggtc gtcgaaattc tacc
//
