LOCUS       Exported                8916 bp ds-DNA     circular SYN 22-DEC-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    LPUtopia-8
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8916)
  AUTHORS   Triple Threat
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, July 25, 2024 from SnapGene 3.2.1
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8916
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_binding    37..84
     misc_recomb     complement(37..84)
                     /note="FRT3"
     misc_feature    113..916
                     /note="AAVS1-HA-L"
     promoter        974..1205
                     /note="core insulin"
     primer_bind     complement(1574..1590)
                     /note="M13 fwd"
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1662..1730
                     /note="LacZ alpha"
     rep_origin      complement(1732..2187)
                     /direction=LEFT
                     /note="F1 ori"
     promoter        2213..2317
                     /gene="bla"
                     /note="AmpR promoter"
     CDS             2318..3178
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      3349..3937
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     polyA_signal    complement(4098..4322)
                     /note="bGH poly(A)"
     CDS             complement(4343..5041)
                     /codon_start=1
                     /product="red fluorescent protein [Acropora millepora] ,  
                     GenBank: AAU06852.1
                     red fluorescent protein FP597 [Acropora millepora], 
                     GenBank: AGI60202.1 and GenBank: ACH89429.1
                     
                     "
                     /note="RFP"
                     /note="Red fluorescent protein, Em(max) 593nm"
                     /db_xref="taxon:<html><body><!--StartFragment-->45264<!--En
                     dFragment--></body></html>"
                     /translation="MALSKHGLTKDMTMKYHMEGSVDGHKFVITGHGNGNPFEGKQTMN
                     LCVVEGGPLPFSEDILSATFDYGNRVFTEYPQGMVDFFKNSCPAGYTWHRSLLFEDGAV
                     CTTSADITVSVEENCFYHNSKFHGVNFPADGPVMKKMTTNWEPSCEKIIPVPRQGILKG
                     DIAMYLLLKDGGRYRCQFDTIYKAKSDPKEMPEWHFIQHKLTREDRSDAKNQKWQLVEH
                     AVASRSALPG"
     promoter        complement(5112..5315)
                     /note="CMV"
     misc_feature    5586..6424
                     /note="AAVS1-HA-R"
     protein_bind    complement(6812..6859)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FRT"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     misc_binding    6812..6859
     polyA_signal    6869..6877
                     /note="bGH poly(A) signal"
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    6897..7244
                     /label=BGHpA
                     /note="BGHpA"
     misc_feature    7079..7097
                     /label=P326
                     /note="P326"
     CDS             complement(7245..7844)
                     /label=PuroR
                     /note="PuroR"
     CDS             complement(7854..7907)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /note="T2A"
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(7908..8624)
     CDS             complement(7908..8624)
                     /codon_start=1
                     /product="enhanced GFP"
                     /note="EGFP"
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     promoter        complement(8669..8687)
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    8687..19
                     /note="Geneious type: promoter uukaryotic"
     promoter        complement(8732..19)
                     /note="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     TATA_signal     8740..8746
ORIGIN
        1 actgccaaaa ccgcatcacg cgccatcgat ctcgacgaag ttcctatact atttgaagaa
       61 taggaacttc ggaataggaa cttcgatatc tgcagaattc gcccttacgc gtgccccact
      121 gtggggtgga ggggacagat aaaagtaccc agaaccagag ccacattaac cggccctggg
      181 aatataaggt ggtcccagct cggggacaca ggatccctgg aggcagcaaa catgctgtcc
      241 tgaagtggac ataggggccc gggttggagg aagaagacta gctgagctct cggacccctg
      301 gaagatgcca tgacaggggg ctggaagagc tagcacagac tagagaggta aggggggtag
      361 gggagctgcc caaatgaaag gagtgagagg tgacccgaat ccacaggaga acggggtgtc
      421 caggcaaaga aagcaagagg atggagaggt ggctaaagcc agggagacgg ggtactttgg
      481 ggttgtccag aaaaacggtg atgatgcagg cctacaagaa ggggaggcgg gacgcaaggg
      541 agacatccgt cggagaaggc catcctaaga aacgagagat ggcacaggcc ccagaaggag
      601 aaggaaaagg gaacccagcg agtgaagacg gcatggggtt gggtgaggga ggagagatgc
      661 ccggagagga cccagacacg gggaggatcc gctcagagga catcacgtgg tgcagcgccg
      721 agaaggaagt gctccggaaa gagcatcctt gggcagcaac acagcagaga gcaaggggaa
      781 gagggagtgg aggaagacgg aacctgaagg aggcggcagg gaaggatctg ggccagccgt
      841 agaggtgacc caggccacaa gctgcagaca gaaagcggca caggcccagg ggagagaatg
      901 ctggtcagag aaagcaggta ccaagggcga attccagcac actggcggcc gttactagta
      961 ggccttggag gccttttccc cgtatccccc caggtgtctg caggctcaaa gagcagcgag
     1021 aagcgttcag aggaaagcga tcccgtgcca ccttccccgt gcccgggctg tccccgcacg
     1081 ctgccggctc ggggatgcgg ggggagcgcc ggaccggagc ggagccccgg gcggctcgct
     1141 gctgccccct agcgggggag ggacgtaatt acatccctgg gggctttggg ggggggctgt
     1201 ccctcataaa agttttgtta ctttatagaa gaaattttga gtttttgttt ttttttaata
     1261 aataaataaa cataaataaa ttgtttgttg aatttattat tagtatgtaa gtgtaaatat
     1321 aataaaactt aatatctatt caaattaata aataaacctc gatatacaga ccgataaaac
     1381 acatgcgtca attttacgca tgattatctt taacgtacgt cacaatatga ttatctttct
     1441 agggttaatc tagctgcgtg ttctgcagcg tgtcgagcat cttcatctgc tccatcacgc
     1501 tgtaaaacac atttgcaccg cgagtctgcc cgtcctccac gggttcaaaa acgtgaatga
     1561 acgaggcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta
     1621 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg
     1681 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct
     1741 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     1801 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     1861 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     1921 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     1981 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     2041 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     2101 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     2161 ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac
     2221 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     2281 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     2341 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     2401 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     2461 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     2521 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     2581 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     2641 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     2701 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     2761 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     2821 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     2881 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     2941 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     3001 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     3061 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     3121 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     3181 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     3241 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     3301 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     3361 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     3421 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     3481 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     3541 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     3601 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     3661 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     3721 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     3781 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     3841 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     3901 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     3961 acggttcctg gccttttctc taggcacccg ttcaattgcc gacccctccc cccaacttct
     4021 cggggactgt gggcgatgtg cgctctgccc actgacgggc accggagcga tcgcagatcc
     4081 ttcgcggccg cagatctcca tagagcccac cgcatcccca gcatgcctgc tattgtcttc
     4141 ccaatcctcc cccttgctgt cctgccccac cccacccccc agaatagaat gacacctact
     4201 cagacaatgc gatgcaattt cctcatttta ttaggaaagg acagtgggag tggcaccttc
     4261 cagggtcaag gaaggcacgg gggaggggca aacaacagat ggctggcaac tagaaggcac
     4321 agggatccga tttaaattcg aattaccctg gcagggcgct acgggatgcc acggcgtgct
     4381 cgaccagttg ccacttctgg ttcttggcat ctgagcggtc ctcgcgggtg agtttgtgct
     4441 ggatgaagtg ccactctggc atctccttgg gatcggactt tgccttgtag atggtatcga
     4501 actggcatcg gtatcttccc ccgtccttga gcaggaggta cattgcgatg tcgcccttca
     4561 agattccctg acgagggacc gggatgattt tctcgcagct gggctcccaa ttcgtagtca
     4621 ttttcttcat cacaggcccg tcggcaggaa agttcacgcc gtggaatttg ctgttgtggt
     4681 agaagcagtt ctcctcgacg gacacggtga tatcagcgga ggtggtacac accgccccgt
     4741 cctcgaagag caggctgcgg tgccaggtgt atcctgcggg acagctgttc ttaaagaaat
     4801 ccaccatacc ctgtggatac tctgtgaaca cgcgattgcc gtagtcaaag gttgcggaca
     4861 ggatgtcctc cgagaagggc aggggtccgc cttccaccac acacaggttc attgtctgtt
     4921 tcccctcgaa tgggttgccg ttcccgtggc ccgtaatcac gaacttgtgc ccgtccaccg
     4981 agccctccat atggtacttc atggtcatgt ccttagtcag gccgtgctta ctaagggcca
     5041 ttctagaatc ttctatggag gtcaaaacag cgtggatggc gtctccaggc gatctgacgg
     5101 ttcactaaac gagctctgct tatataaacc tcccaccgta cacgcctacc gcccatttgc
     5161 gtcaatgggg cggagttgtt acgacatttt ggaaagtccc gttgattttg gtgccaaaac
     5221 aaactcccat tgacgtcaat ggggtggaga cttggaaatc cccgtgagtc aaaccgctat
     5281 ccacgcccat tgatgtactg ccaaaaccgc atcaccatgg taatagcgat gactaatacg
     5341 tagatgtact gccaagtagg aaagtcccat aaggtcatgt actgggcata attcatcctc
     5401 ggcaaactct ttccatgccg cttcaacctc gcgggaaaag gcacgggctt cttcctcccc
     5461 gatgcccaga tagcgccagc ttgggcgatg actgagccgg aaaaaagacc cgacgatatg
     5521 atcctgatgc agctagatta accctagaaa gatagtctgc gtaaaattga cgcatgcatc
     5581 tcgagagagc agagccagga acccctgtag ggaaggggca ggagagccag gggcatgaga
     5641 tggtggacga ggaaggggga cagggaagcc tgagcgcctc tcctgggctt gccaaggact
     5701 caaacccaga agcccagagc agggccttag ggaagcggga ccctgctctg ggcggaggaa
     5761 tatgtcccag atagcactgg ggactcttta aggaaagaag gatggagaaa gagaaaggga
     5821 gtagaggcgg ccacgacctg gtgaacacct aggacgcacc attctcacaa agggagtttt
     5881 ccacacggac acccccctcc tcaccacagc cctgccagga cggggctggc tactggcctt
     5941 atctcacagg taaaactgac gcacggagga acaatataaa ttggggacta gaaaggtgaa
     6001 gagccaaagt tagaactcag gaccaactta ttctgatttt gtttttccaa actgcttctc
     6061 ctcttgggaa gtgtaaggaa gctgcagcac caggatcagt gaaacgcacc agacagccgc
     6121 gtcagagcag ctcaggttct gggagagggt agcgcagggt ggccactgag aaccgggcag
     6181 gtcacgcatc ccccccttcc ctcccacccc ctgccaagct ctccctccca ggatcctctc
     6241 tggctccatc gtaagcaaac cttagaggtt ctggcaagga gagagatggc tccaggaaat
     6301 gggggtgtgt caccagataa ggaatctgcc taacaggagg tgggggttag acccaatatc
     6361 aggagactag gaaggaggag gcctaaggat ggggcttttc tgtcaccaat cctgtcccta
     6421 gtagtgtacg ttaaagataa tcatgcgtaa aattgacgca tgtgttttat cggtctgtat
     6481 atcgaggttt atttattaat ttgaatagat attaagtttt attatattta cacttacata
     6541 ctaataataa attcaacaaa caatttattt atgtttattt atttattaaa aaaaaacaaa
     6601 aactcaaaat ttcttctata aagtaacaaa acttttaaac attctctctt ttacaaaaat
     6661 aaacttattt tgtactttaa aaacagtcat gttgtattat aaaataagta attagcttaa
     6721 cctatacata atagaaacaa attatactta ttagtcagtc agaaacaact ttggcacata
     6781 tcaatattat gctctcgtta attaactcga ggaagttcct atactttcta gagaatagga
     6841 acttcggaat aggaacttcg tcgacaagcc atagagcggt ggttacgcgc taaagctgct
     6901 attgtcttcc caatcctccc ccttgctgtc ctgccccacc ccacccccca gaatagaatg
     6961 acacctactc agacaatgcg atgcaatttc ctcattttat taggaaagga cagtgggagt
     7021 ggcaccttcc agggtcaagg aaggcacggg ggaggggcaa acaacagatg gctggcaact
     7081 agaaggcaca gtcgaggctg atcagcgggt ttaaacgggc cctctagagt cggtgggcct
     7141 cgggggcggg tgcggggtcg gcggggccgc cccgggtggc ttcggtcgga gccatggggt
     7201 cgtgcgctcc tttcggtcgg gcgctgcggg tcgtggggcg ggcgtcaggc accgggcttg
     7261 cgggtcatgc accaggtgcg cggtccttcg ggcacctcga cgtcggcggt gacggtgaag
     7321 ccgagccgct cgtagaaggg gaggttgcgg ggcgcggagg tctccaggaa ggcgggcacc
     7381 ccggcgcgct cggccgcctc cactccgggg agcacgacgg cgctgcccag acccttgccc
     7441 tggtggtcgg gcgagacgcc gacggtggcc aggaaccacg cgggctcctt gggccggtgc
     7501 ggcgccagga ggccttccat ctgttgctgc gcggccagcc gggaaccgct caactcggcc
     7561 atgcgcgggc cgatctcggc gaacaccgcc cccgcttcga cgctctccgg cgtggtccag
     7621 accgccaccg cggcgccgtc gtccgcgacc cacaccttgc cgatgtcgag cccgacgcgc
     7681 gtgaggaaga gttcttgcag ctcggtgacc cgctcgatgt ggcggtccgg gtcgacggtg
     7741 tggcgcgtgg cggggtagtc ggcgaacgcg gcggcgaggg tgcgtacggc ccgggggacg
     7801 tcgtcgcggg tggcgaggcg caccgtgggc ttgtactcgg tcatttcgag cctagggccg
     7861 ggattctcct ccacgtcacc gcatgttaga agacttcctc tgccctcctt gtacagctcg
     7921 tccatgccga gagtgatccc ggcggcggtc acgaactcca gcaggaccat gtgatcgcgc
     7981 ttctcgttgg ggtctttgct cagggcggac tgggtgctca ggtagtggtt gtcgggcagc
     8041 agcacggggc cgtcgccgat gggggtgttc tgctggtagt ggtcggcgag ctgcacgctg
     8101 ccgtcctcga tgttgtggcg gatcttgaag ttcaccttga tgccgttctt ctgcttgtcg
     8161 gccatgatat agacgttgtg gctgttgtag ttgtactcca gcttgtgccc caggatgttg
     8221 ccgtcctcct tgaagtcgat gcccttcagc tcgatgcggt tcaccagggt gtcgccctcg
     8281 aacttcacct cggcgcgggt cttgtagttg ccgtcgtcct tgaagaagat ggtgcgctcc
     8341 tggacgtagc cttcgggcat ggcggacttg aagaagtcgt gctgcttcat gtggtcgggg
     8401 tagcggctga agcactgcac gccgtaggtc agggtggtca cgagggtggg ccagggcacg
     8461 ggcagcttgc cggtggtgca gatgaacttc agggtcagct tgccgtaggt ggcatcgccc
     8521 tcgccctcgc cggacacgct gaacttgtgg ccgtttacgt cgccgtccag ctcgaccagg
     8581 atgggcacca ccccggtgaa cagctcctcg cccttgctca ccatggtggc gaccggtgga
     8641 tccgagctcg gtaccaagct tgggtctccc tatagtgagt cgtattaatt tcgataagcc
     8701 agtaagcagt gggttctcta gttagccaga gagctctgct tatatagacc tcccaccgta
     8761 cacgcctacc gcccatttgc gtcaatgggg cggagttgtt acgacatttt ggaaagtccc
     8821 gttgattttg gtgccaaaac aaactcccat tgacgtcaat ggggtggaga cttggaaatc
     8881 cccgtgagtc aaaccgctat ccacgcccat tgatgt
//
