LOCUS       sequence-393204-        7970 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  piggyBAC vector backbone with GFP for cloning DNA Ticker Tapes and 
            U6 for pegRNAs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7970)
  TITLE     Molecular recording using DNA Typewriter
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7970)
  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            "Unpublished"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: piggyBAC-U6-GFP-WPRE
FEATURES             Location/Qualifiers
     source          1..7970
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..105)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(131..586)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(268..289)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     480..499
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     713..735
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     727..744
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     728..744
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   874..936
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        1361..1609
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1361..1381
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1532..1551
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        1792..2970
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2023..2961
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2918..2938
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      2977..2986
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2983..2985,2986..2988,2989..3702)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICITGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAVVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3028..3049)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3289..3308)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3636..3657
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3800..4388
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             complement(4271..4282)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4540..4661
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4577..4596)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4631..4650
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5424..5458
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5949..5965)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5949..5965)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5962..5984)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5973..5989
                     /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(5997..6003,6004..6021,6022..6027))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6042..6063
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6180..6197)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6351..6939)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6431..6450)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7110..7901,7902..7970))
                     /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     7733..7752
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      121 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
      181 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
      241 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
      301 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
      361 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
      421 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
      481 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
      541 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc
      601 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
      661 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
      721 cgacgttgta aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg
      781 gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg
      841 ctcgacacgc tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg
      901 tacgttaaag ataatcatgt gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag
      961 gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata
     1021 ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaac aaaaactcaa
     1081 aatttcttct ataaagtaac aaaactttta tgagggacag ccccccccca aagcccccag
     1141 ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc
     1201 cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg
     1261 ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga
     1321 cacctggggg gatacgggga aaaggcctcc acggccaaga gagggcctat ttcccatgat
     1381 tccttcatat ttgcatatac gatacaaggc tgttagagag ataattagaa ttaatttgac
     1441 tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta
     1501 gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa
     1561 gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg gagacggttg
     1621 taaatttgag acattggtgg acgcgtcgtc tcattttttt attacaggga cagcagagat
     1681 ccactttggc gccggctcga gggggcccgg gtgcaaagat ggataaagtt ttaaacagag
     1741 aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat tggctccggt
     1801 gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc
     1861 ggcaattgat ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg
     1921 tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc
     1981 gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt
     2041 tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccacc
     2101 tggctgcagt acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg
     2161 aggccttgcg cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc
     2221 tggggccgcc gcgtgcgaac ctggtggcac cttcgcgcct gtctcgctgc tttcgataag
     2281 tctctagcca tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt
     2341 cttgtaaatg cgggccaaga tctgcacact ggtatttcgg tttttggggc cgcgggcggc
     2401 gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca
     2461 ccgagaatcg gacgggggta gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg
     2521 ccgccgtgta tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga
     2581 gcggaaagat ggccgcttcc cggccctgct gcagggagct caaaatggag gacgcggcgc
     2641 tcgggagagc gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc
     2701 gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg
     2761 agcttttgga gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc
     2821 cacactgagt gggtggagac tgaagttagg ccagcttggc acttgatgta attctccttg
     2881 gaatttgccc tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa
     2941 agtttttttc ttccatttca ggtgtcgtga cgtacggcca ccatggtgag caagggcgag
     3001 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
     3061 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag
     3121 ttcatctgca tcaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc
     3181 tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag
     3241 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
     3301 tacaagaccc gcgccgtggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
     3361 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac
     3421 aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc
     3481 aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
     3541 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc
     3601 gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     3661 gccgccggga tcactctcgg catggacgag ctgtacaagt gaagtcgact ttccaagctc
     3721 cataggtctg atttggtacc atagcagatg atccatggta ccgctttaag gccggtccta
     3781 gcaatcgcgt taagtcgaca atcaacctct ggattacaaa atttgtgaaa gattgactgg
     3841 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     3901 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     3961 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     4021 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
     4081 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     4141 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc
     4201 gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg
     4261 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
     4321 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
     4381 ctccccgcgt cgactttaag accaatgact tacaaggcac gcgtaatcaa cctctggatt
     4441 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
     4501 gatacgctgc tttaatgcct ttgtatcatg cgttaactaa acttgtttat tgcagcttat
     4561 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     4621 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gaattgactc
     4681 aaatgatgtc aattagtcta tcagaagctc atctggtctc ccttccgggg gacaagacat
     4741 ccctgtttaa tatttaaaca gcagtgttcc caaactgggt tcttatatcc cttgctctgg
     4801 tcaaccaggt tgcagggttt cctgtcctca caggaacgaa gtccctaaag aaacagtggc
     4861 agccaggttt agccccggaa ttgactggat tcctttttta gggcccattg gtatggcttt
     4921 ttccccgtat ccccccaggt gtctgcaggc tcaaagagca gcgagaagcg ttcagaggaa
     4981 agcgatcccg tgccaccttc cccgtgcccg ggctgtcccc gcacgctgcc ggctcgggga
     5041 tgcgggggga gcgccggacc ggagcggagc cccgggcggc tcgctgctgc cccctagcgg
     5101 gggagggacg taattacatc cctgggggct ttgggggggg gctgtccctg atatctataa
     5161 caagaaaata tatatataat aagttatcac gtaagtagaa catgaaataa caatataatt
     5221 atcgtatgag ttaaatctta aaagtcacgt aaaagataat catgcgtcat tttgactcac
     5281 gcggtcgtta tagttcaaaa tcagtgacac ttaccgcatt gacaagcacg cctcacggga
     5341 gctccaagcg gcgactgaga tgtcctaaat gcacagcgac ggattcgcgc tatttagaaa
     5401 gagagagcaa tatttcaaga atgcatgcgt caattttacg cagactatct ttctagggtt
     5461 aatctagctg catcaggatc atatcgtcgg gtcttttttc cggctcagtc atcgcccaag
     5521 ctggcgctat ctgggcatcg gggaggaaga agcccgtgcc ttttcccgcg aggttgaagc
     5581 ggcatggaaa gagtttgccg aggatgactg ctgctgcatt gacgttgagc gaaaacgcac
     5641 gtttaccatg atgattcggg aaggtgtggc catgcacgcc tttaacggtg aactgttcgt
     5701 tcaggccacc tgggatacca gttcgtcgcg gcttttccgg acacagttcc ggatggtcag
     5761 cccgaagcgc atcagcaacc cgaacaatac cggcgacagc cggaactgcc gtgccggtgt
     5821 gcagattaat gacagcggtg cggcgctggg atattacgtc agcgaggacg ggtatcctgg
     5881 ctggatgccg cagaaatgga catggatacc ccgtgagtta cccggcgggc gcgcttggcg
     5941 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     6001 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     6061 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     6121 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     6181 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     6241 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     6301 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     6361 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     6421 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     6481 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     6541 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     6601 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     6661 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     6721 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     6781 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     6841 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     6901 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     6961 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     7021 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     7081 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     7141 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     7201 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     7261 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     7321 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     7381 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     7441 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     7501 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     7561 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     7621 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     7681 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     7741 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     7801 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     7861 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     7921 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
//
