LOCUS       sequence-460317-        6028 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A puromycin selectable, PiggyBac destination vector compatible with 
            4-way multi-site gateway cloning system .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6028)
  AUTHORS   Gillespie WB 3rd, Zhang Y, Ruiz OE, Cerda J 3rd, Ortiz-Guzman J, 
            Sherman M, Turner WD, Largoza G, Mosser LE, Fujimoto E, Chien CB, 
            Kwan KM, Arenkiel BR, Devine WP, Wythe JD
  TITLE     MultiSite Assembly of Gateway Induced Clones (MAGIC): a flexible 
            cloning toolbox for use in vertebrate model systems.
  JOURNAL   Development. 2025 Nov 15;152(22):dev204308. doi: 10.1242/dev.204308.
            Epub 2025 Nov 13.
  PUBMED    41230856
REFERENCE   2  (bases 1 to 6028)
  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: 
            "Development."; date: "2025-11-15"; volume: "152(22):dev204308. 
            doi"; pages: " 10.1242/dev.204308. Epub 2025 Nov 13"
COMMENT     Sequence Label: pB-Puro-DEST (JDW 925)
FEATURES             Location/Qualifiers
     source          1..6028
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        24..128
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(129..197,198..989)
                     /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     complement(347..366)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1160..1748
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1649..1668
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1921..1938
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     1945..1961
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   1987..2049
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    2252..2375
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(2416..2721)
                     /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
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(2518..2537)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(3041..3721)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     3636..3655
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(3775..3781,3782..3799,3800..3805))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(3830..3953)
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3961..3977)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3986..4002)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3987..4003)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        4023..4339
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4190..4325
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4252..4271
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4387..4434
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4453..5052
                     /codon_start=1
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(5092..5111)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5136..5184
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     complement(5400..5421)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     repeat_region   5705..5739
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     5919..5938
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
       61 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
      121 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
      181 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
      241 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
      301 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
      361 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
      421 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
      481 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
      541 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
      601 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
      661 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
      721 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
      781 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
      841 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
      901 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
      961 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     1021 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     1081 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     1141 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     1201 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     1261 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     1321 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     1381 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     1441 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     1501 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     1561 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     1621 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     1681 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     1741 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     1801 ctcacatgct gtacagctta agtcatgttc tttcctgcgt tatcccctga ttctgtggat
     1861 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     1921 agcgagtcag tgagcgagga agctgtaaaa cgacggccag tagctccacc gccgcagcta
     1981 gattaaccct agaaagataa tcatattgtg acgtacgtta aagataatca tgcgtaaaat
     2041 tgacgcatgt gttttatcgg tctgtatatc gaggtttatt tattaatttg aatagatatt
     2101 aagttttatt atatttacac ttacatacta ataataaatt caacaaacaa tttatttatg
     2161 tttatttatt tattaaaaaa aaacaaaaac tcaaaatttc ttctataaag taacaaaact
     2221 tttatgacta gtcatgatta cgccaagcta tcaactttgt atagaaaagt tgaacgagaa
     2281 acgtaaaatg atataaatat caatatatta aattagattt tgcataaaaa acagactaca
     2341 taatactgta aaacacaaca tatccagtca ctatggtcga cctgcagact ggctgtgtat
     2401 aagggagcct gacatttata ttccccagaa catcaggtta atggcgtttt tgatgtcatt
     2461 ttcgcggtgg ctgagatcag ccacttcttc cccgataacg gagaccggca cactggccat
     2521 atcggtggtc atcatgcgcc agctttcatc cccgatatgc accaccgggt aaagttcacg
     2581 ggggacttta tctgacagca gacgtgcact ggccaggggg atcaccatcc gtcgcccggg
     2641 cgtgtcaata atatcactct gtacatccac aaacagacga taacggctct ctcttttata
     2701 ggtgtaaacc ttaaactgca tttcaccagc ccctgttctc gtcggcaaag gagccgttca
     2761 tttcaataaa ccgggcgacc tcagccatcc cttcctgatt ttccgctttc cagcgttcgg
     2821 cacgcagacg acgggcttca ttctgcatgg ttgtgcttac cgaaccggag atattgacat
     2881 catatatgcc ttgagcaact gatagctgtc gctgtcaact gtcactgtaa tacgctgctt
     2941 catagcatac ctctttttga catacttcgg gtatacatat cagtatatat tcttataccg
     3001 caaaaatcag cgcgcaaata cgcatactgt tatctggctt ttagtaagcc ggatcctcta
     3061 gattacgccc cgcctgccac tcatcgcagt actgttgtaa ttcattaagc attctgccga
     3121 catggaagcc atcacaaacg gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt
     3181 cgccttgcgt ataatatttg cccatggtga aaacgggggc gaagaagttg tccatattgg
     3241 ccacgtttaa atcaaaactg gtgaaactca cccagggatt ggctgagacg aaaaacatat
     3301 tctcaataaa ccctttaggg aaataggcca ggttttcacc gtaacacgcc acatcttgcg
     3361 aatatatgtg tagaaactgc cggaaatcgt cgtggtattc actccagagc gatgaaaacg
     3421 tttcagtttg ctcatggaaa acggtgtaac aagggtgaac actatcccat atcaccagct
     3481 caccgtcttt cattgccata cggaattccg gatgagcatt catcaggcgg gcaagaatgt
     3541 gaataaaggc cggataaaac ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa
     3601 tatccagctg aacggtctgg ttataggtac attgagcaac tgactgaaat gcctcaaaat
     3661 gttctttacg atgccattgg gatatatcaa cggtggtata tccagtgatt tttttctcca
     3721 ttttagcttc cttagctcct gaaaatctcg acggatccta actcaaaatc cacacattat
     3781 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgcggccgcc atagtgactg
     3841 gatatgttgt gttttacagt attatgtagt ctgtttttta tgcaaaatct aatttaatat
     3901 attgatattt atatcatttt acgtttctcg ttcaacttta ttatacatag ttgataattc
     3961 actggccgtc gttttacggt accatcgata ccgtcgacct cgacatggtc atagctgttg
     4021 acggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     4081 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     4141 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     4201 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     4261 agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg
     4321 aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca
     4381 gcacgtgttg acaattaatc atcggcatag tatatcggca tagtataata cgacaaggtg
     4441 aggaactaaa ccatgaccga gtacaagcct accgtgcgcc tggccactcg cgatgatgtg
     4501 ccccgcgccg tccgcactct ggccgccgct ttcgccgact accccgctac ccggcacacc
     4561 gtggaccccg accggcacat cgagcgtgtg acagagttgc aggagctgtt cctgacccgc
     4621 gtcgggctgg acatcggcaa ggtgtgggta gccgacgacg gcgcggccgt ggccgtgtgg
     4681 actacccccg agagcgttga ggccggcgcc gtgttcgccg agatcggccc ccgaatggcc
     4741 gagctgagcg gcagccgcct ggccgcccag cagcaaatgg agggcctgct tgccccccat
     4801 cgtcccaagg agcctgcctg gtttctggcc actgtaggag tgagccccga ccaccagggc
     4861 aagggcttgg gcagcgccgt cgtgttgccc ggcgtagagg ccgccgaacg cgccggtgtg
     4921 cccgcctttc tcgaaacaag cgcaccaaga aaccttccat tctacgagcg cctgggcttc
     4981 accgtgaccg ccgatgtcga ggtgcccgag ggacctagga cctggtgtat gacacgaaaa
     5041 cctggcgcct aacgtaccgg ttagtaatga gtttaaacgg gggaggctaa ctgaaacacg
     5101 gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac
     5161 gcacgggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc tggcactctg
     5221 tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc ttttccccac
     5281 cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc
     5341 ctgccatagc agatccggtt aaatcaaaag aatagaccga gatagggttg agtgttgttc
     5401 cagtttggaa caagagtcca cgctgtccct gatatctata acaagaaaat atatatataa
     5461 taagttatca cgtaagtaga acatgaaata acaatataat tatcgtatga gttaaatctt
     5521 aaaagtcacg taaaagataa tcatgcgtca ttttgactca cgcggtcgtt atagttcaaa
     5581 atcagtgaca cttaccgcat tgacaagcac gcctcacggg agctccaagc ggcgactgag
     5641 atgtcctaaa tgcacagcga cggattcgcg ctatttagaa agagagagca atatttcaag
     5701 aatgcatgcg tcaattttac gcagactatc tttctagggt taatctagct gcatcaaggg
     5761 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     5821 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     5881 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     5941 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     6001 cttaatgcgc cgctacaggg cgcgtcag
//
