LOCUS       sequence-152800-        6450 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector containing a cassette for scarless insertion of sfGFP and 
            marked by the visible marker 3xP3-DsRed flanked by piggyBac termini.
            .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6450)
  TITLE     Kate O'Connor-Giles Lab Donor Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6450)
  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            "Unpublished"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: pScarlessHD-sfGFP-DsRed
FEATURES             Location/Qualifiers
     source          1..6450
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(11..28)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(182..770)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(262..281)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             join(894..962,963..1754)
                     /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(1112..1131)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             complement(2004..2798)
                     /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(2004..2798)
                     /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(2115..2134)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2725..2744
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(3147..3449)
                     /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="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(3249..3268)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     3572..3594
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3586..3603
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3587..3603
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3610..3629
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3610..3628
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   4421..4483
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     regulatory      4878..4887
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4884..5561
                     /codon_start=1
                     /product="rapidly maturing tetrameric variant of DsRed 
                     fluorescent protein (Bevis and Glick, 2002)"
                     /label=DsRed-Express
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAEGRHH
                     LFL"
     primer_bind     complement(5014..5032)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(5064..5084)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     5282..5301
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     5476..5499
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    5682..5803
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5719..5738)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5773..5792
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   6078..6112
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(6191..6209)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6230..6246)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6230..6246)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6243..6265)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6254..6270
                     /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(6278..6284,6285..6302,6303..6308))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6323..6344
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
       61 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
      121 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
      181 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      241 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      301 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      361 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      421 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      481 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      541 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
      601 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
      661 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
      721 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
      781 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
      841 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
      901 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
      961 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     1021 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     1081 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     1141 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     1201 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     1261 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     1321 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     1381 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     1441 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     1501 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     1561 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     1621 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     1681 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     1741 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     1801 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgagcggat
     1861 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     1921 aagtgccacc tgatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     1981 aggaaattgt aagcgttaat aattcagaag aactcgtcaa gaaggcgata gaaggcgatg
     2041 cgctgcgaat cgggagcggc gataccgtaa agcacgagga agcggtcagc ccattcgccg
     2101 ccaagctctt cagcaatatc acgggtagcc aacgctatgt cctgatagcg gtccgccaca
     2161 cccagccggc cacagtcgat gaatccagaa aagcggccat tttccaccat gatattcggc
     2221 aagcaggcat cgccatgggt cacgacgaga tcctcgccgt cgggcatgct cgccttgagc
     2281 ctggcgaaca gttcggctgg cgcgagcccc tgatgctctt cgtccagatc atcctgatcg
     2341 acaagaccgg cttccatccg agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg
     2401 aatgggcagg tagccggatc aagcgtatgc agccgccgca ttgcatcagc catgatggat
     2461 actttctcgg caggagcaag gtgagatgac aggagatcct gccccggcac ttcgcccaat
     2521 agcagccagt cccttcccgc ttcagtgaca acgtcgagca cagctgcgca aggaacgccc
     2581 gtcgtggcca gccacgatag ccgcgctgcc tcgtcttgca gttcattcag ggcaccggac
     2641 aggtcggtct tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa cacggcggca
     2701 tcagagcagc cgattgtctg ttgtgcccag tcatagccga atagcctctc cacccaagcg
     2761 gccggagaac ctgcgtgcaa tccatcttgt tcaatcatgc gaaacgatcc tcatcctgtc
     2821 tcttgatcag agcttgatcc cctgcgccat cagatccttg gcggcaagaa agccatccag
     2881 tttactttgc agggcttccc aaccttacca gagggcgccc cagctggcaa ttccggttcg
     2941 cttgctgtcc ataaaaccgc ccagtctagc tatcgccatg taagcccact gcaagctacc
     3001 tgctttctct ttgcgcttgc gttttccctt gtccagatag cccagtagct gacattcatc
     3061 cggggtcagc accgtttctg cggactggct ttctacgtga aaaggatcta ggtgaagatc
     3121 ctttttgata atctcatgcc tgacatttat attccccaga acatcaggtt aatggcgttt
     3181 ttgatgtcat tttcgcggtg gctgagatca gccacttctt ccccgataac ggagaccggc
     3241 acactggcca tatcggtggt catcatgcgc cagctttcat ccccgatatg caccaccggg
     3301 taaagttcac gggagacttt atctgacagc agacgtgcac tggccagggg gatcaccatc
     3361 cgtcgccccg gcgtgtcaat aatatcactc tgtacatcca caaacagacg ataacggctc
     3421 tctcttttat aggtgtaaac cttaaactgc cgtacgtata ggctgcgcaa ctgttgggaa
     3481 gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca
     3541 aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc
     3601 agtgaattgt aatacgactc actatagggc gaattgaatt tagcggccgc gaattcgccc
     3661 ttggtggatc tggaggttcc ggcggctcag ggggtagtat ggtgtccaag ggcgaggagc
     3721 tgttcaccgg cgtggtgccc atcctggtgg agctggatgg cgacgtgaac ggccacaagt
     3781 tcagcgtgcg cggcgagggc gagggcgacg ccaccaacgg caagctgacc ctgaagttca
     3841 tctgcaccac cggcaagctg cccgtgccct ggcccaccct ggtgaccacc ctgacctacg
     3901 gcgtgcagtg cttcagccgc taccccgatc acatgaagca gcacgatttc ttcaagagcg
     3961 ccatgcccga gggctacgtg caggagcgca ccatcagctt caaggatgac ggcacctaca
     4021 agacccgcgc cgaggtgaag ttcgagggcg ataccctggt gaaccgcatc gagctgaagg
     4081 gcatcgattt caaggaggat ggcaacatcc tgggccacaa gctggagtac aacttcaaca
     4141 gccacaacgt gtacatcacc gccgataagc agaagaacgg catcaaggcc aacttcaaga
     4201 tccgccacaa tgtggaggat ggctccgtgc agctggccga tcactaccag cagaacaccc
     4261 ccatcggcga cggcccagtg ctgctgcccg ataaccacta cctgagcacc cagagcgtgc
     4321 tgtccaagga ccccaacgag aagcgcgatc acatggtgct gctggagttc gtgaccgccg
     4381 ccggcatcac cctgggcatg gatgagctgt acaagattaa ccctagaaag ataatcatat
     4441 tgtgacgtac gttaaagata atcatgcgta aaattgacgc atgtgtttta tcggtctgta
     4501 tatcgaggtt tatttattaa tttgaataga tattaagttt tattatattt acacttacat
     4561 actaataata aattcaacaa acaatttatt tatgtttatt tatttattaa aaaaaaacaa
     4621 aaactcaaaa tttcttctat aaagtaacaa aacttttagg atctaattca attagagact
     4681 aattcaatta gagctaattc aattaggatc caagcttatc gatttcgaac cctcgaccgc
     4741 cggagtataa atagaggcgc ttcgtctacg gagcgacaat tcaattcaaa caagcaaagt
     4801 gaacacgtcg ctaagcgaaa gctaagcaaa taaacaagcg cagctgaaca agctaaacaa
     4861 tcggctcgaa gccggtcgcc accatggcct cctccgagga cgtcatcaag gagttcatgc
     4921 gcttcaaggt gcgcatggag ggctccgtga acggccacga gttcgagatc gagggcgagg
     4981 gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggcggcc
     5041 ccctgccctt cgcctgggac atcctgtccc cccagttcca gtacggctcc aaggtgtacg
     5101 tgaagcaccc cgccgacatc cccgactaca agaagctgtc cttccccgag ggcttcaagt
     5161 gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc
     5221 tccaggacgg ctccttcatc tacaaggtga agttcatcgg cgtgaacttc ccctccgacg
     5281 gccccgtaat gcagaagaag actatgggct gggaggcgtc caccgagcgc ctgtaccccc
     5341 gcgacggcgt gctgaagggc gagatccaca aggccctgaa gctgaaggac ggcggccact
     5401 acctggtgga gttcaagtcc atctacatgg ccaagaagcc cgtgcagctg cccggctact
     5461 actacgtgga ctccaagctg gacatcacct cccacaacga ggactacacc atcgtggagc
     5521 agtacgagcg cgccgagggc cgccaccacc tgttcctgta gcggccgcga ctctagatca
     5581 taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc
     5641 ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt
     5701 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     5761 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttagatatct ataacaagaa
     5821 aatatatata taataagtta tcacgtaagt agaacatgaa ataacaatat aattatcgta
     5881 tgagttaaat cttaaaagtc acgtaaaaga taatcatgcg tcattttgac tcacgcggtc
     5941 gttatagttc aaaatcagtg acacttaccg cattgacaag cacgcctcac gggagctcca
     6001 agcggcgact gagatgtcct aaatgcacag cgacggattc gcgctattta gaaagagaga
     6061 gcaatatttc aagaatgcat gcgtcaattt tacgcagact atctttctag ggttaaggcc
     6121 ggcgggtcgg gaggttctgg tggttcagga ggttccaagg gcgaattcgt ttaaacctgc
     6181 aggactagtc cctttagtga gggttaattc tgagcttggc gtaatcatgg tcatagctgt
     6241 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     6301 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     6361 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     6421 cggggagagg cggtttgcgt attgggcgct
//
