LOCUS       pKG3560_pHarbor-        4958 bp    DNA     circular SYN 07-MAY-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 4958)
  AUTHORS   ISU Biology IT
  TITLE     Direct Submission
  JOURNAL   Exported May 7, 2025 from SnapGene 8.0.3
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4958
                     /mol_type="other DNA"
                     /organism="recombinant plasmid"
     primer_bind     1..40
                     /label=oTA465 fix_lp_bb_fwd
     primer_bind     complement(1..40)
                     /label=oTA468 fix_lp_in_rev
     primer_bind     complement(2..19)
                     /label=M13fwd (#10 & #176)
     primer_bind     21..45
                     /label=oTA240 PuroRepeat-bb-frag_fwd
     promoter        31..135
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(68..89)
                     /label=oSEQ095 oKL157
     CDS             join(136..204,205..996)
                     /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     136..160
                     /label=oSEQ083 Amp-fwd
     primer_bind     complement(354..373)
                     /label=oSEQ066 Amp_rev
     primer_bind     complement(397..416)
                     /label=Amp_seq3 (#119)
     primer_bind     complement(623..634)
                     /label=oTA467 fix_lp_in_fwd
     primer_bind     complement(997..1016)
                     /label=Amp_seq1 (#120)
     rep_origin      1167..1755
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1656..1675
                     /label=oSEQ067 pBR322-ori_fwd
     primer_bind     complement(1807..1852)
                     /label=oTA466 fix_lp_bb_rev
     primer_bind     complement(1811..1832)
                     /label=oTA233 PuroRepeat-bb-frag_rev
     primer_bind     1813..1850
                     /label=oTA467 fix_lp_in_fwd
     promoter        1839..3016
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     primer_bind     complement(1899..1916)
                     /label=oSEQ045 phage-dest-rev-2
     primer_bind     complement(2803..2824)
                     /label=oSEQ046 phage-dest-upstream-seq
     primer_bind     2810..2832
                     /label=EF1a_qPCRfwd (#195)
     primer_bind     2867..2886
                     /label=oSEQ064 EF1a_fwd
     primer_bind     complement(2901..2923)
                     /label=EF1a_qPCRrev (#196)
     misc_recomb     3034..3075
                     /label=attB
                     /note="from myobacteriumphage bxb1 (Thomson et al 2012)"
     primer_bind     3082..3167
                     /label=Harbor-F-NFKB-loxP
     primer_bind     3082..3162
                     /label=loxP-BbsI-F-repair
     primer_bind     3082..3160
                     /label=attR1-F-loxP
     primer_bind     3082..3159
                     /label=pHA-loxP-F-BbsI
     primer_bind     3082..3159
                     /label=pHA-loxP-F-BbsIa
     protein_bind    3082..3115
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     primer_bind     3082..3114
                     /label=loxP-F
     primer_bind     3082..3111
                     /label=loxP-BbsI-F-repaira
     primer_bind     complement(3083..3119)
                     /label=loxP-F
     primer_bind     3107..3159
                     /label=pKG01835-FWD
     primer_bind     complement(3116..3140)
                     /label=neo4 (#13)
     primer_bind     3119..3165
                     /label=DP-309
     primer_bind     complement(3123..3140)
                     /label=DP-312
     primer_bind     3126..3169
                     /label=DP-305
     primer_bind     3141..3172
                     /label=oHA012 blast_harbor_internal_fwd
     primer_bind     3141..3164
                     /label=LentiX1-harbor-3to5-fwd
     primer_bind     3141..3164
                     /label=LentiX1-harbor-5to3-fwd
     primer_bind     3141..3162
                     /label=oTA236 PuroRepeat-Harbor_frag_fwd
     primer_bind     3141..3160
                     /label=indel-bb-F
     protein_bind    3141
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     primer_bind     3156..3185
                     /label=oTA153 aavs_puro_R1_fwd
     primer_bind     3158..3194
                     /label=min-lenti-dest-fwd
     primer_bind     3164..3185
                     /label=Harbor-F-Rogi1HAup
     protein_bind    3173..3297
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3174..3198)
                     /label=oSEQ049 attR1-rev
     primer_bind     complement(3174..3198)
                     /label=?oSEQ049 attR1-rev
     primer_bind     3178..3204
                     /label=oTA170 pb_R2_rev
     primer_bind     3281..3321
                     /label=oTA155 dest-ccdB_fwd
     primer_bind     complement(3281..3321)
                     /label=oTA154 R1_d-ccdB_rev
     primer_bind     3284..3298
                     /label=Primer 2
     primer_bind     3302..3317
                     /label=oGE036 gg_entry_fwd
     promoter        join(3330..3335,3336..3353,3354..3360)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     primer_bind     3346..3371
                     /label=Primer 1
     CDS             3414..4094
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     complement(3832..3863)
                     /label=oTA134 gg_internal_rev
     primer_bind     3832..3863
                     /label=oTA135 gg_internal_fwd
     primer_bind     complement(3940..3959)
                     /label=CmR-R-ligation
     primer_bind     3960..3979
                     /label=CmR-F
     primer_bind     complement(3960..3979)
                     /label=CmR-R
     CDS             4414..4719
                     /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."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     4598..4616
                     /label=oSEQ012 ccdB_for
     primer_bind     4692..4761
                     /label=oTA157 single_int_R2_fwd
     primer_bind     complement(4722..4769)
                     /label=Primer 2
     primer_bind     complement(4724..4743)
                     /label=oSEQ019 SEQ_junk_check_for
     primer_bind     complement(4732..4744)
                     /label=oTA150 gg_paqc1_entry_rev
     primer_bind     complement(4755..4772)
                     /label=oTA155 dest-ccdB_fwd
     primer_bind     4755..4772
                     /label=oTA154 R1_d-ccdB_rev
     protein_bind    complement(4756..4880)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4849..4885)
                     /label=oTA170 pb_R2_rev
     primer_bind     complement(4859..4881)
                     /label=INT-Track-pCW-rev
     primer_bind     complement(4860..4881)
                     /label=Harbor-R-Rogi1HAdown
     primer_bind     complement(4861..4880)
                     /label=min-lenti-dest-rev
     primer_bind     complement(4865..4895)
                     /label=oHA029 PB_harbor_internal_rev
     primer_bind     complement(4871..4930)
                     /label=Harbor-R-NFKB-lox2772
     primer_bind     complement(4876..4930)
                     /label=attR2-R-lox2772
     primer_bind     complement(4878..4895)
                     /label=LentiX1-harbor-5to3-rev
     primer_bind     complement(4878..4895)
                     /label=LentiX1-harbor-3to5-rev
     primer_bind     complement(4878..4895)
                     /label=DP-306
     primer_bind     complement(4879..4938)
                     /label=pHA-R-lox2772
     primer_bind     complement(4883..4895)
                     /label=oTA237 PuroRepeat-Harbor_frag_rev
     primer_bind     4895..4925
                     /label=loxP-BbsI-F-repaira
     protein_bind    4896..4929
                     /label=lox2272
                     /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 (Lee and 
                     Saito, 1988)."
     primer_bind     4896..4928
                     /label=loxP-F
     primer_bind     complement(4897..4930)
                     /label=loxP-F
     primer_bind     complement(join(4936..4958,1..17))
                     /label=DP-310
     primer_bind     join(4954..4958,1..13)
                     /label=DP-311
     primer_bind     join(4955..4958,1..12)
                     /label=DP-471
ORIGIN
        1 cactggccgt cgttttacaa ctataatgtg cgcggaaccc ctatttgttt atttttctaa
       61 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
      121 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
      181 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
      241 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
      301 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
      361 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
      421 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
      481 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
      541 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
      601 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
      661 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
      721 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
      781 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
      841 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
      901 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
      961 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     1021 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     1081 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     1141 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     1201 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     1261 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     1321 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     1381 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     1441 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     1501 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     1561 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     1621 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     1681 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     1741 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     1801 ccttttgctc acatgttctt tcctgcgtta tcgaattggc tccggtgccc gtcagtgggc
     1861 agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaaccgg
     1921 tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct
     1981 ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt
     2041 tcgcaacggg tttgccgcca gaacacaggt aagtgccgtg tgtggttccc gcgggcctgg
     2101 cctctttacg ggttatggcc cttgcgtgcc ttgaattact tccacctggc tgcagtacgt
     2161 gattcttgat cccgagcttc gggttggaag tgggtgggag agttcgaggc cttgcgctta
     2221 aggagcccct tcgcctcgtg cttgagttga ggcctggcct gggcgctggg gccgccgcgt
     2281 gcgaatctgg tggcaccttc gcgcctgtct cgctgctttc gataagtctc tagccattta
     2341 aaatttttga tgacctgctg cgacgctttt tttctggcaa gatagtcttg taaatgcggg
     2401 ccaagatctg cacactggta tttcggtttt tggggccgcg ggcggcgacg gggcccgtgc
     2461 gtcccagcgc acatgttcgg cgaggcgggg cctgcgagcg cggccaccga gaatcggacg
     2521 ggggtagtct caagctggcc ggcctgctct ggtgcctggc ctcgcgccgc cgtgtatcgc
     2581 cccgccctgg gcggcaaggc tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc
     2641 gcttcccggc cctgctgcag ggagctcaaa atggaggacg cggcgctcgg gagagcgggc
     2701 gggtgagtca cccacacaaa ggaaaagggc ctttccgtcc tcagccgtcg cttcatgtga
     2761 ctccacggag taccgggcgc cgtccaggca cctcgattag ttctcgagct tttggagtac
     2821 gtcgtcttta ggttgggggg aggggtttta tgcgatggag tttccccaca ctgagtgggt
     2881 ggagactgaa gttaggccag cttggcactt gatgtaattc tccttggaat ttgccctttt
     2941 tgagtttgga tcttggttca ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc
     3001 atttcaggtg tcgtgaggaa ttcgccacca tggccggctt gtcgacgacg gcggtctccg
     3061 tcgtcaggat catccggatc cataacttcg tatagcatac attatacgaa gttatcatga
     3121 tattcggcaa gcaggcatcg tactcatagc aggtgccact agtggagttc cgacaagttt
     3181 gtacaaaaaa gctgaacgag aaacgtaaaa tgatataaat atcaatatat taaattagat
     3241 tttgcataaa aaacagacta cataatactg taaaacacaa catatccagt cactatggcg
     3301 gattcgcggc cgcattaggc accccaggct ttacacttta tgcttccggc tcgtataatg
     3361 tgtggatttt gagttaggat ccgtcgagat tttcaggagc taaggaagct aaaatggaga
     3421 aaaaaatcac tggatatacc acagttgata tatcccaatg gcatcgtaaa gaacattttg
     3481 aggcatttca gtcagttgct caatgtacct ataaccagac cgttcagctg gatattacgg
     3541 cctttttaaa gaccgtaaag aaaaataagc acaagtttta tccggccttt attcacattc
     3601 ttgcccgcct gatgaatgct catccggaat tccgtatggc aatgaaagac ggtgagctgg
     3661 tgatatggga tagtgttcac ccttgttaca ccgttttcca tgagcaaact gaaacgtttt
     3721 catcgctctg gagtgaatac cacgacgatt tccggcagtt tctacacata tattcgcaag
     3781 atgtggcgtg ttacggtgaa aacctggcct atttccctaa agggtttatt gagaatatgt
     3841 ttttcgtttc agccaatccc tgggtgagtt tcaccagttt tgatttaaac gtggccaata
     3901 tggacaactt cttcgccccc gttttcacca tgggcaaata ttatacgcaa ggcgacaagg
     3961 tgctgatgcc gctggcgatt caggttcatc atgccgtttg tgatggcttc catgtcggca
     4021 gaatgcttaa tgaattacaa cagtactgcg atgagtggca ggcggggcgt aatctagagg
     4081 atccggctta ctaaaagcca gataacagta tgcgtatttg cgcgctgatt tttgcggtat
     4141 aagaatatat actgatatgt atacccgaag tatgtcaaaa agaggtatgc tatgaagcag
     4201 cgtattacag tgacagttga cagcgacagc tatcagttgc tcaaggcata tatgatgtca
     4261 atatctccgg tctggtaagc acaaccatgc agaatgaagc ccgtcgtctg cgtgccgaac
     4321 gctggaaagc ggaaaatcag gaagggatgg ctgaggtcgc ccggtttatt gaaatgaacg
     4381 gctcttttgc tgacgagaac aggggctggt gaaatgcagt ttaaggttta cacctataaa
     4441 agagagagcc gttatcgtct gtttgtggat gtacagagtg atattattga cacgcccggg
     4501 cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt cccccgtgaa
     4561 ctttacccgg tggtgcatat cggggatgaa agctggcgca tgatgaccac cgatatggcc
     4621 agtgtgccgg tttccgttat cggggaagaa gtggctgatc tcagccaccg cgaaaatgac
     4681 atcaaaaacg ccattaacct gatgttctgg ggaatataaa tgtcaggctc ccttatacac
     4741 agcctgcagg tcgaccatag tgactggata tgttgtgttt tacagtatta tgtagtctgt
     4801 tttttatgca aaatctaatt taatatattg atatttatat cattttacgt ttctcgttca
     4861 gctttcttgt acaaagtggt cacctgctat gcgctataac ttcgtatagg atactttata
     4921 cgaagttatc accagagacc gtccggggtc tctgcgct
//
