LOCUS       Exported                6245 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  HMEJ donor for fusing a p2A-mCherry reporter to mouse Cdx2. AAV 
            backbone..
ACCESSION   .
VERSION     .
KEYWORDS    AAV_Cdx2 HMEJ donor
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6245)
  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 6245)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6245
                     /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             complement(794..805)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1069..1125
                     /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             1126..1836
                     /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(1271..1289)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1539..1558
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2678..2697)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2679..2697)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2704..2721)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2704..2720)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2713..2735)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2861..3289
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2948..2967)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3158..3179
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             3633..4427
                     /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(3687..3706)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4297..4316
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             4445..5305
                     /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(4663..4682)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5476..6064
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5965..5984
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6218..6235
                     /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 gtaccagacc acgggagggg tcactgggtg attgcagggg acacccagtg cctctggctt
      301 ctgtggatat atatgaatgt atacagacat acacatatca aagatttaaa aatccttgaa
      361 cttttttttt cattaacaca aaagaggccc tacttgcaca ccaacacttt ctgtagtcta
      421 gggaaaaggg aaggaacaaa caagagaaat tcatagccag gaagttttgg acaaagtttc
      481 attgtctgtg ccaaggtgaa gaacatcgca gtaccaatga gctgtgggag cccaagggtg
      541 acccacgagg cacactgagc tggccaacca tttgtgtagg gaatagcttt cgtcatggtt
      601 ccgttccctg gttctgaggt tctgttgcta gtaaagggtt ttttgtttgt ttgttttttg
      661 ttttttgaaa gcaacttggg gagggggcag gtaagtaaag aggacgaggc tggcctggct
      721 ttgggaacat ttcccaaact cagtgaaaga aggacgctcc cggggtggcg ccatttctct
      781 gttcttatct tcttctacct tctattttgt catctctagg ttaaaatttg gtttcagaac
      841 cgcagagcca aggagaggaa aatcaagaag aagcagcagc agcaacagca gcagcagcaa
      901 caacagcctc cacagccgcc gccacaacct tcccagcctc agccgggtgc cctgcggagc
      961 gtgcccgagc ccttgagtcc tgtgacctcc ttgcaaggct cagtgcctgg ttctgtccct
     1021 ggggttctgg ggccagctgg aggggtttta aactccactg tcacccaggc cactaacttc
     1081 tccctgttga aacaagcagg ggatgtcgaa gagaatcccg ggccaatggt gagcaagggc
     1141 gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca catggagggc
     1201 tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc
     1261 acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc
     1321 ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc
     1381 gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag
     1441 gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac
     1501 aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc
     1561 atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag
     1621 atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt caagaccacc
     1681 tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat caagttggac
     1741 atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc cgagggccgc
     1801 cactccaccg gcggcatgga cgagctgtac aagtaagacc cctcccgtgg tctgaagcgg
     1861 cggcggcaca gcaatcccag gctgagccat gaggagtatg gacgctgcga gaatcctcag
     1921 aagagattcc tctcctccta cccacgaaca gcatctactg atggagattg aggacagaag
     1981 atgagtggaa ttatggacct caggggaaga catggtttag attttttttt tctttttaac
     2041 ttttcccatt ccgactcttc ctgccagcaa cgacaaacga agtgattcct ggggcttctt
     2101 cgttcatgct ctttgccagg actgactacc gacatgaagc tatcagcctc ttttgcccca
     2161 gctctttgcc tctctgtatt tctgtgtgga gctgaggaga gagtgagact ggatggggtg
     2221 ggggtagcaa tacttgagcc aaggtggctg tttcctgctg actgctttct gagaaccagc
     2281 tggccgtcct gcctccgggc cagggactat tcaaactaca ggagccagag gcagctaaga
     2341 tagctggact gaccgaagtc tgcagaacct cccccaccag gtggtctggg ctttcttctc
     2401 cacaaatcag gaaggggtgg tgggttcagg ggctgcggtg agagggggtt ggttagccaa
     2461 cgccaggccc ctgcgacaag ggcttgttta gaaagcctgt caccagagct gctgtaggcg
     2521 gaatgtatgt ctgtgttgta aatgccagag ccaacctgga cttcctgtcc cttccctcgt
     2581 ctttggctga agaagaccgg aattgtttgc tgctgttcga gtcactgatc tgtgtaccca
     2641 gtgacccctc ccgtggtctt ctagagggcc caattcgccc tatagtgagt cgtattacaa
     2701 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     2761 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     2821 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg acgcgccctg tagcggcgca
     2881 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     2941 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     3001 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     3061 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     3121 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     3181 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     3241 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaatt
     3301 cagggcgcaa gggctgctaa aggaagcgga acacgtagaa agccagtccg cagaaacggt
     3361 gctgaccccg gatgaatgtc agctactggg ctatctggac aagggaaaac gcaagcgcaa
     3421 agagaaagca ggtagcttgc agtgggctta catggcgata gctagactgg gcggttttat
     3481 ggacagcaag cgaaccggaa ttgccagctg gggcgccctc tggtaaggtt gggaagccct
     3541 gcaaagtaaa ctggatggct ttcttgccgc caaggatctg atggcgcagg ggatcaagat
     3601 ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag
     3661 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     3721 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     3781 agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc
     3841 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     3901 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatcccac cttgctcctg
     3961 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     4021 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     4081 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     4141 tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg
     4201 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     4261 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     4321 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     4381 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgaatt gaaaaaggaa
     4441 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     4501 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     4561 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     4621 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     4681 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     4741 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     4801 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     4861 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     4921 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     4981 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     5041 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     5101 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     5161 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     5221 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     5281 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     5341 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     5401 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     5461 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     5521 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     5581 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     5641 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     5701 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     5761 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     5821 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     5881 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     5941 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     6001 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     6061 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     6121 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     6181 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     6241 ggaag
//
