LOCUS       AAVS1-Bxb1-LP-v2        8259 bp DNA     circular SYN 10-NOV-2022
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 8259)
  AUTHORS   Trial User
  TITLE     Direct Submission
  JOURNAL   Exported Nov 10, 2022 from SnapGene 6.1.1
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8259
                     /mol_type="other DNA"
                     /organism="recombinant plasmid"
     misc_feature    33..836
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
                     /note="color: #ffe4c4"
     misc_feature    874..893
                     /label=MCS
                     /note="MCS"
                     /note="color: #0080ff"
     protein_bind    894..926
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
                     /note="color: #31849b; direction: RIGHT"
     promoter        982..1481
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
                     /note="color: #ffffff; direction: RIGHT"
     CDS             1496..2212
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /note="This feature has 3 segments:
                        1: 1496 .. 1498 / #3366ff
                        2: 1499 .. 1501 / #3366ff / 1a
                        3: 1502 .. 2212 / #3366ff"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     CDS             2220..2222
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /note="color: #05fd14"
                     /translation=""
     polyA_signal    2265..2386
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
                     /note="color: #a6acb3"
     protein_bind    2405..2452
                     /label=attP
                     /bound_moiety="bxb1 integrase"
                     /note="color: #31849b; direction: RIGHT"
     CDS             2458..2826
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /note="color: #ccffcc"
                     /translation="AKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDDV
                     TLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGRE
                     FALRDPAGNCVHFVAEEQD"
     polyA_signal    2835..3357
                     /label=polyA
                     /note="Rabbit bGlobulin polyAd signal"
                     /note="color: #ff0080"
     protein_bind    3419..3451
                     /label=lox251
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites."
                     /note="color: #31849b; direction: RIGHT"
     misc_feature    3495..4331
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
                     /note="color: #ffe4c4"
     promoter        complement(4366..4384)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /note="color: #ffffff; direction: LEFT"
     CDS             4545..4847
                     /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."
                     /note="color: #993366"
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     CDS             complement(6244..7104)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="This feature has 2 segments:
                        1: 6244 .. 7035 / #ccffcc
                        2: 7036 .. 7104 / #ccffcc / signal sequence
                     Cleavage site after base 7035"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      7228..7816
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     promoter        8140..8170
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
                     /note="This forward directional feature has 3 segments:
                        1: 8140 .. 8145 / #ffffff / -35
                        2: 8146 .. 8163 / #ffffff
                        3: 8164 .. 8170 / #ffffff / -10"
     protein_bind    8178..8194
                     /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)."
                     /note="color: #31849b"
     promoter        8239..8257
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
                     /note="color: #ffffff; direction: RIGHT"
ORIGIN
        1 ctagtcctgc aggtttaaac gaattcgccc tttgctttct ctgaccagca ttctctcccc
       61 tgggcctgtg ccgctttctg tctgcagctt gtggcctggg tcacctctac ggctggccca
      121 gatccttccc tgccgcctcc ttcaggttcc gtcttcctcc actccctctt ccccttgctc
      181 tctgctgtgt tgctgcccaa ggatgctctt tccggagcac ttccttctcg gcgctgcacc
      241 acgtgatgtc ctctgagcgg atcctccccg tgtctgggtc ctctccgggc atctctcctc
      301 cctcacccaa ccccatgccg tcttcactcg ctgggttccc ttttccttct ccttctgggg
      361 cctgtgccat ctctcgtttc ttaggatggc cttctccgac ggatgtctcc cttgcgtccc
      421 gcctcccctt cttgtaggcc tgcatcatca ccgtttttct ggacaacccc aaagtacccc
      481 gtctccctgg ctttagccac ctctccatcc tcttgctttc tttgcctgga caccccgttc
      541 tcctgtggat tcgggtcacc tctcactcct ttcatttggg cagctcccct acccccctta
      601 cctctctagt ctgtgctagc tcttccagcc ccctgtcatg gcatcttcca ggggtccgag
      661 agctcagcta gtcttcttcc tccaacccgg gcccctatgt ccacttcagg acagcatgtt
      721 tgctgcctcc agggatcctg tgtccccgag ctgggaccac cttatattcc cagggccggt
      781 taatgtggct ctggttctgg gtacttttat ctgtcccctc caccccacag tggggcaagg
      841 tccagtcacg acgttgtaaa acgacggcca gtcgctagcg agctcggcgc gccataactt
      901 cgtatagcat acattatacg aagttagaat tcgccttctg caggagcgta cagaacccag
      961 ggccctggca cccgtgctac cgggtagggg aggcgctttt cccaaggcag tctggagcat
     1021 gcgctttagc agccccgctg ggcacttggc gctacacaag tggcctctgg cctcgcacac
     1081 attccacatc caccggtagg cgccaaccgg ctccgttctt tggtggcccc ttcgcgccac
     1141 cttccactcc tcccctagtc aggaagttcc cccccgcccc gcagctcgcg tcgtgcagga
     1201 cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca gatggacagc accgctgagc
     1261 aatggaagcg ggtaggcctt tggggcagcg gccaatagca gctttgctcc ttcgctttct
     1321 gggctcagag gctgggaagg ggtgggtccg ggggcgggct caggggcggg ctcaggggcg
     1381 gggcgggcgc ccgaaggtcc tccggaggcc cggcattctg cacgcttcaa aagcgcacgt
     1441 ctgccgcgct gttctcctct tcctcatctc cgggcctttc gacctgcagg ccaccatggt
     1501 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     1561 cgtaaacggc cacaagttca gcgtgagggg cgagggcgag ggcgatgcca ccaacggcaa
     1621 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     1681 gaccaccctg agccacggcg tgcagtgctt cgcccgctac cccgaccaca tgaagcagca
     1741 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     1801 ggacgacggc acctacaaga cccgcgccga ggtgaagttc gagggcgaca ccctagtgaa
     1861 ccgcatcgag ctgaagggcg tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     1921 ggagtacaac ttcaacagcc acaacatcta tatcatggcc gtcaagcaga agaacggcat
     1981 caaggtgaac ttcaagatcc gccacaacgt ggaggacggc agcgtgcagc tcgccgacca
     2041 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca gccactacct
     2101 gagcacccag tccgtgctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     2161 ggagttccgc accgccgccg ggatcactct cggcatggac gagctgtaca agtaatcgat
     2221 aactgcagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     2281 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     2341 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctagatat
     2401 acgtggtttg tctggtcaac caccgcggtc tcagtggtgt acggtacaaa cccatatgcc
     2461 aagttgacca gtgccgttcc ggtgctcacc gcgcgcgacg tcgccggagc ggtcgagttc
     2521 tggaccgacc ggctcgggtt ctcccgggac ttcgtggagg acgacttcgc cggtgtggtc
     2581 cgggacgacg tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc ggacaacacc
     2641 ctggcctggg tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc ggaggtcgtg
     2701 tccacgaact tccgggacgc ctccgggccg gccatgaccg agatcggcga gcagccgtgg
     2761 gggcgggagt tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt ggccgaggag
     2821 caggactgac attcactcct caggtgcagg ctgcctatca gaaggtggtg gctggtgtgg
     2881 ccaatgccct ggctcacaaa taccactgag atctttttcc ctctgccaaa aattatgggg
     2941 acatcatgaa gccccttgag catctgactt ctggctaata aaggaaattt attttcattg
     3001 caatagtgtg ttggaatttt ttgtgtctct cactcggaag gacatatggg agggcaaatc
     3061 atttaaaaca tcagaatgag tatttggttt agagtttggc aacatatgcc catatgctgg
     3121 ctgccatgaa caaaggtggc tataaagagg tcatcagtat atgaaacagc cccctgctgt
     3181 ccattcctta ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt
     3241 tgtgttattt ttttctttaa catccctaaa attttcctta catgttttac tagccagatt
     3301 tttcctcctc tcctgactac tcccagtcat agctgtccct cttctcttat gaagatcgga
     3361 tcgctactag aggatgcaca tgtgaccgag ggaggagctg caagaactct tcctcacgat
     3421 aacttcgtat aggagacttt atacgaagtt aagcgcttcg tgagaccacg aagtggctct
     3481 tcagtggacg aattactagg gacaggattg gtgacagaaa agccccatcc ttaggcctcc
     3541 tccttcctag tctcctgata ttgggtctaa cccccacctc ctgttaggca gattccttat
     3601 ctggtgacac acccccattt cctggagcca tctctctcct tgccagaacc tctaaggttt
     3661 gcttacgatg gagccagaga ggatcctggg agggagagct tggcaggggg tgggagggaa
     3721 gggggggatg cgtgacctgc ccggttctca gtggccaccc tgcgctaccc tctcccagaa
     3781 cctgagctgc tctgacgcgg ctgtctggtg cgtttcactg atcctggtgc tgcagcttcc
     3841 ttacacttcc caagaggaga agcagtttgg aaaaacaaaa tcagaataag ttggtcctga
     3901 gttctaactt tggctcttca cctttctagt ccccaattta tattgttcct ccgtgcgtca
     3961 gttttacctg tgagataagg ccagtagcca gccccgtcct ggcagggctg tggtgaggag
     4021 gggggtgtcc gtgtggaaaa ctccctttgt gagaatggtg cgtcctaggt gttcaccagg
     4081 tcgtggccgc ctctactccc tttctctttc tccatccttc tttccttaaa gagtccccag
     4141 tgctatctgg gacatattcc tccgcccaga gcagggtccc gcttccctaa ggccctgctc
     4201 tgggcttctg ggtttgagtc cttggcaagc ccaggagagg cgctcaggct tccctgtccc
     4261 ccttcctcgt ccaccatctc atgcccctgg ctctcctgcc ccttccctac aggggttcct
     4321 ggctctgctc taagggcgaa ttcgcggccg ctaaattcaa ttcgccctat agtgagtcgt
     4381 attacaattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
     4441 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
     4501 gcaccgatcg cccttcccaa cagttgcgca gcctatacgt acggcagttt aaggtttaca
     4561 cctataaaag agagagccgt tatcgtctgt ttgtggatgt acagagtgat attattgaca
     4621 cgccggggcg acggatggtg atccccctgg ccagtgcacg tctgctgtca gataaagtct
     4681 cccgtgaact ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg
     4741 atatggccag tgtgccggtc tccgttatcg gggaagaagt ggctgatctc agccaccgcg
     4801 aaaatgacat caaaaacgcc attaacctga tgttctgggg aatataaatg tcaggcatga
     4861 gattatcaaa aaggatcttc acctagatcc ttttcacgta gaaagccagt ccgcagaaac
     4921 ggtgctgacc ccggatgaat gtcagctact gggctatctg gacaagggaa aacgcaagcg
     4981 caaagagaaa gcaggtagct tgcagtgggc ttacatggcg atagctagac tgggcggttt
     5041 tatggacagc aagcgaaccg gaattgccag ctggggcgcc ctctggtaag gttgggaagc
     5101 cctgcaaagt aaactggatg gctttcttgc cgccaaggat ctgatggcgc aggggatcaa
     5161 gctctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg
     5221 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa
     5281 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg
     5341 tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt
     5401 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa
     5461 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc
     5521 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg
     5581 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg
     5641 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg
     5701 aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg
     5761 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     5821 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     5881 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     5941 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgaattatt
     6001 aacgcttaca atttcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     6061 cgcatcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     6121 aatacattca aatatgtatc cgctcatgag attatcaaaa aggatcttca cctagatcct
     6181 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     6241 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     6301 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     6361 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     6421 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     6481 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     6541 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     6601 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     6661 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     6721 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     6781 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     6841 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     6901 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     6961 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     7021 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     7081 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     7141 gggttattgt ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     7201 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     7261 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     7321 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     7381 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     7441 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     7501 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     7561 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     7621 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     7681 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     7741 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     7801 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     7861 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     7921 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     7981 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     8041 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     8101 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
     8161 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
     8221 tgattacgcc aagctcagaa ttaaccctca ctaaaggga
//
