LOCUS       Exported                6268 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  HMEJ donor for fusing a p2A-mCherry reporter to mouse Nanog. AAV 
            backbone..
ACCESSION   .
VERSION     .
KEYWORDS    AAV_Nanog HMEJ donor
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6268)
  AUTHORS   Yao X, Wang X, Hu X, Liu Z, Liu J, Zhou H, Shen X, Wei Y, Huang Z, 
            Ying W, Wang Y, Nie YH, Zhang CC, Li S, Cheng L, Wang Q, Wu Y, Huang
            P, Sun Q, Shi L, Yang H
  TITLE     Homology-mediated end joining-based targeted integration using 
            CRISPR/Cas9.
  JOURNAL   Cell Res. 2017 May 19. doi: 10.1038/cr.2017.76.
  PUBMED    28524166
REFERENCE   2  (bases 1 to 6268)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6268
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        143..173
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1084..1140
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1141..1851
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1286..1304)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1554..1573
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2701..2720)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2702..2720)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2727..2744)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2727..2743)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2736..2758)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2884..3312
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2971..2990)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3181..3202
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             3656..4450
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3710..3729)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4320..4339
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             4468..5328
                     /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(4686..4705)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5499..6087
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5988..6007
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6241..6258
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttg
      241 gtacccgtaa gtctcatatt tcacctggcc ccctccatct taccatctta ttttatgtgt
      301 ctgtgccgtg tgtgtgtgtg tgtgtgtgtg tgtggctgac ctatggctgt tgggtatgga
      361 tcccagattt cctgtaagag cagtcagtgg ctctgccttt gcttcccagt gtcatcctga
      421 tccatctatg tccattagtc ctgtcccttt cccatccctc ttcctccacg cactctttct
      481 tgtgatcttt ggttctttga ggcaggctct ctctatgtag ccctggcagt cagagatctg
      541 cctgcctagt ctcaggagtg ctggggttaa cggcctgtgc ggccatgcta atcttttcag
      601 tatttctgca gtttacatct ttagagtccc cttttcactc attgcattaa tcacctcttt
      661 cagaagggct cagcaccagt ggagtatccc agcatccatt gcagctatcc ccagggctat
      721 ctggtgaacg catctggaag cctttccatg tggggcagcc agacttggac caacccaact
      781 tggagcagcc agacctggac caacccaact tggaacaacc agacctggac caacccaact
      841 tggagcagcc aggcctggac cgctcagtcc tggaacggcc agccttggaa tgctgctccg
      901 ctccataact tcggggagga ctttctgcag ccttacgtac agttgcagca aaacttctct
      961 gccagtgatt tggaggtgaa tttggaagcc actagggaaa gccatgcgca ttttagcacc
     1021 ccacaagcct tggaattatt cctgaactac tctgtgactc caccaggtga aatattaatt
     1081 aacgccacta acttctccct gttgaaacaa gcaggggatg tcgaagagaa tcccgggcca
     1141 atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag
     1201 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc
     1261 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc
     1321 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac
     1381 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc
     1441 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac
     1501 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta
     1561 atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc
     1621 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct
     1681 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc
     1741 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa
     1801 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagta aggcgcgccg
     1861 aaatatgaga cttacgcaac atctgggctt aaagtcaggg caaagccagg ttccttcctt
     1921 cttccaaata ttttcatatt ttttttaaag atttatttat tcattatatg taagtacact
     1981 gtagctgtct tcagacactc cagaagaggg cgtcagatct tgttacgtat ggttgtgagc
     2041 caccatgtgg ttgctgggat ttgaactcct gaccttcgga agagcagtcg ggtgctctta
     2101 tccactgagc catctcacca gcccctggtt tattttttta attattattt gctttttgtt
     2161 tatcaagaca gggtttctct gcatagctct aattgtcttt gaactagctc tgcagaccag
     2221 cctggccttg aactcagaga tctgcccact tatctttgcc tcctgaatgc tgggaccaaa
     2281 ggtggcatac caccacacct ggcatatata ttgtttattt ctatttctat ttttattggt
     2341 gccagagcaa acctaggact tagaacatgc tgggcaccaa ctcaacttct gagctctatt
     2401 tacaacttgg tgtgttagtg tatttgtctt agttctgaat ttgtcctttt tttagtgtta
     2461 actctaggct ttggagacag tgaggtgcat atactctctc cttcccaaga ataagtgctt
     2521 gaacaccctt acccacgccc acccacccat gctagtcttt tttcttagaa gcgtgggtct
     2581 tggtatacac tgtgtcattt tgaggggtga ggtttaaaag tatatacaaa gtataacgat
     2641 atggtggcta ctctcgaggc caggtgaaat atgagactta cgtctagagg gcccaattcg
     2701 ccctatagtg agtcgtatta caattcactg gccgtcgttt tacaacgtcg tgactgggaa
     2761 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     2821 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     2881 tggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     2941 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     3001 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     3061 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     3121 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     3181 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     3241 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     3301 ttaacgcgaa ttttaacaaa attcagggcg caagggctgc taaaggaagc ggaacacgta
     3361 gaaagccagt ccgcagaaac ggtgctgacc ccggatgaat gtcagctact gggctatctg
     3421 gacaagggaa aacgcaagcg caaagagaaa gcaggtagct tgcagtgggc ttacatggcg
     3481 atagctagac tgggcggttt tatggacagc aagcgaaccg gaattgccag ctggggcgcc
     3541 ctctggtaag gttgggaagc cctgcaaagt aaactggatg gctttcttgc cgccaaggat
     3601 ctgatggcgc aggggatcaa gatctgatca agagacagga tgaggatcgt ttcgcatgat
     3661 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     3721 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     3781 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga
     3841 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     3901 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     3961 cctgtcatcc caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     4021 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     4081 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     4141 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga
     4201 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     4261 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     4321 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     4381 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     4441 gttcttctga attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     4501 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     4561 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     4621 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     4681 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     4741 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     4801 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     4861 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     4921 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     4981 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     5041 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     5101 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     5161 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     5221 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     5281 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     5341 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     5401 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     5461 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     5521 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     5581 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     5641 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     5701 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     5761 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     5821 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     5881 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     5941 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     6001 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     6061 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     6121 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     6181 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     6241 agcgagtcag tgagcgagga agcggaag
//
