LOCUS       sequence-478738-        6024 bp    DNA     circular SYN 16-JUL-2025
DEFINITION  A PiggyBac destination vector compatible with gateway with 2 core 
            cHS4 insulators by each ITR site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6024)
  AUTHORS   Gillespie W, Zhang Y, Ruiz OE, Cerda J, Ortiz-Guzman J, Turner WD, 
            Largoza G, Sherman M, 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 with diverse applications in vertebrate model 
            systems.
  JOURNAL   bioRxiv [Preprint]. 2024 Jul 13:2024.07.13.603267. doi: 
            10.1101/2024.07.13.603267.
  PUBMED    39026881
REFERENCE   2  (bases 1 to 6024)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 16, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2024 Jul 13:2024.07.13.603267. doi: 
            10.1101/2024.07.13.603267."
COMMENT     Sequence Label: pB-2x-lns-DEST (JDW 1206)
FEATURES             Location/Qualifiers
     source          1..6024
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   1066..1128
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     regulatory      1931..1940
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        join(1960..1965,1966..1983,1984..1990)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             2044..2724
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITTFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     complement(2110..2129)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             3044..3349
                     /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     3228..3247
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(3390..3513)
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     repeat_region   4312..4346
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4399..4416)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4399..4415)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4408..4430)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4624..4643)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4743..4765
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4803..4821)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4889..4993
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4994..5062,5063..5854)
                     /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(5212..5231)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctcg cttgcacgcc
     1021 tctgcactcg tcggtcccgg catccgattc tagatacgta tttaacccta gaaagataat
     1081 catattgtga cgtacgttaa agataatcat gcgtaaaatt gacgcatgtg ttttatcggt
     1141 ctgtatatcg aggtttattt attaatttga atagatatta agttttatta tatttacact
     1201 tacatactaa taataaattc aacaaacaat ttatttatgt ttatttattt attaaaaaaa
     1261 aacaaaaact caaaatttct tctataaagt aacaatacgt aactagttca gcctaaagct
     1321 ttttccccgt atccccccag gtgtctgcag gctcaaagag cagcgagaag cgttcagagg
     1381 aaagcgatcc cgtgccacct tccccgtgcc cgggctgtcc ccgcacgctg ccggctcggg
     1441 gatgcggggg gagcgccgga ccggagcgga gccccgggcg gctcgctgct gccccctagc
     1501 gggggaggga cgtaattaca tccctggggg ctttgggggg gggctgtccc cgtgcactca
     1561 gcctaaagct ttttccccgt atccccccag gtgtctgcag gctcaaagag cagcgagaag
     1621 cgttcagagg aaagcgatcc cgtgccacct tccccgtgcc cgggctgtcc ccgcacgctg
     1681 ccggctcggg gatgcggggg gagcgccgga ccggagcgga gccccgggcg gctcgctgct
     1741 gccccctagc gggggaggga cgtaattaca tccctggggg ctttgggggg gggctgtccc
     1801 ggtaccaatt atcaactatg tataaaaagt tgaacgagaa acgtaaaatg atataaatat
     1861 caatatatta aattagattt tgcataaaaa acagactaca taatactgta aaacacaaca
     1921 tatccagtca ccatggcggc cgcattaggc agcccaggct ttacacttta tgcttccggc
     1981 tcgtataatg tgtggatttt gagttaggat ccgtcgagat tttcaggagc taaggaagct
     2041 aaaatggaga aaaaaatcac tggatatacc accgttgata tatcccaatg gcatcgtaaa
     2101 gaacattttg aggcatttca gtcagttgct caatgtacct ataaccagac cgttcagctg
     2161 gatattacga cctttttaaa gaccgtaaag aaaaataagc acaagtttta tccggccttt
     2221 attcacattc ttgcccgcct gatgaatgct catccggaat tccgtatggc aatgaaagac
     2281 ggtgagctgg tgatatggga tagtgttcac ccttgttaca ccgttttcca tgagcaaact
     2341 gaaacgtttt catcgctctg gagtgaatac cacgacgatt tccggcagtt tctacacata
     2401 tattcgcaag atgtggcgtg ttacggtgaa aacctggcct atttccctaa agggtttatt
     2461 gagaatatgt ttttcgtctc agccaatccc tgggtgagtt tcaccagttt tgatttaaac
     2521 gtggccaata tggacaactt cttcgccccc gttttcacca tgggcaaata ttatacgcaa
     2581 ggcgacaagg tgctgatgcc gctggcgatt caggttcatc atgccgtttg tgatggcttc
     2641 catgtcggca gaatgcttaa tgaattacaa cagtactgcg atgagtggca ggcggggcgt
     2701 aatctagagg atccggctta ctaaaagcca gataacagta tgcgtatttg cgcgctgatt
     2761 tttgcggtat aagaatatat actgatatgt atacccgaag tatgtcaaaa agaggtatgc
     2821 tatgaagcag cgtattacag tgacagttga cagcgacagc tatcagttgc tcaaggcata
     2881 tatgatgtca atatctccgg ttcggtaagc acaaccatgc agaatgaagc ccgtcgtctg
     2941 cgtgccgaac gctggaaagc ggaaaatcag gaggggatgg ctgaggtcgc ccggtttatt
     3001 gaaatgaacg gctcctttgc cgacgagaac aggggctggt gaaatgcagt ttaaggttta
     3061 cacctataaa agagagagcc gttatcgtct gtttgtggat gtacagagtg atattattga
     3121 cacgcccggg cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt
     3181 cccccgtgaa ctttacccgg tggtgcatat cggggatgaa agctggcgca tgatgaccac
     3241 cgatatggcc agtgtgccgg tctccgttat cggggaagaa gtggctgatc tcagccaccg
     3301 cgaaaatgac atcaaaaacg ccattaacct gatgttctgg ggaatataaa tgtcaggctc
     3361 ccttatacac agccagtctg caggtcgacc atagtgactg gatatgttgt gttttacagt
     3421 attatgtagt ctgtttttta tgcaaaatct aatttaatat attgatattt atatcatttt
     3481 acgtttctcg ttcaactttt ctatacaaag ttgatagctt ggcgtaatca tggtcgactc
     3541 agcctaaagc tttttccccg tatcccccca ggtgtctgca ggctcaaaga gcagcgagaa
     3601 gcgttcagag gaaagcgatc ccgtgccacc ttccccgtgc ccgggctgtc cccgcacgct
     3661 gccggctcgg ggatgcgggg ggagcgccgg accggagcgg agccccgggc ggctcgctgc
     3721 tgccccctag cgggggaggg acgtaattac atccctgggg gctttggggg ggggctgtcc
     3781 ccgtgcactc agcctaaagc tttttccccg tatcccccca ggtgtctgca ggctcaaaga
     3841 gcagcgagaa gcgttcagag gaaagcgatc ccgtgccacc ttccccgtgc ccgggctgtc
     3901 cccgcacgct gccggctcgg ggatgcgggg ggagcgccgg accggagcgg agccccgggc
     3961 ggctcgctgc tgccccctag cgggggaggg acgtaattac atccctgggg gctttggggg
     4021 ggggctgtcc cctcgaggat atctataaca agaaaatata tatataataa gttatcacgt
     4081 aagtagaaca tgaaataaca atataattat cgtatgagtt aaatcttaaa agtcacgtaa
     4141 aagataatca tgcgtcattt tgactcacgc ggtcgttata gttcaaaatc agtgacactt
     4201 accgcattga caagcacgcc tcacgggagc tccaagcggc gactgagatg tcctaaatgc
     4261 acagcgacgg attcgcgcta tttagaaaga gagagcaata tttcaagaat gcatgcgtca
     4321 attttacgca gactatcttt ctagggttaa gatatcggat ccatctcgac gcattcgcga
     4381 agtaccgatc tccaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
     4441 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
     4501 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct
     4561 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatct ggtgcactct
     4621 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc
     4681 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     4741 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa
     4801 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     4861 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     4921 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     4981 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     5041 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     5101 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     5161 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     5221 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     5281 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     5341 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     5401 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     5461 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     5521 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     5581 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     5641 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     5701 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     5761 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     5821 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     5881 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     5941 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     6001 cgtagaaaag atcaaaggat cttc
//
